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PubMed1997

Biovar diversity is reflected by variations of genes encoding urease of Ureaplasma urealyticum.

Five oligonucleotide primers derived from the gene encoding urease of Ureaplasma urealyticum were designed to evaluate the relationship between the urease gene and biovar diversity of this organism. Five combinations of these primers were tested by PCR and the result revealed that there were variations in urease genes among different serovars of U. urealyticum. This result, in agreement with other PCRs based on other functionally unrelated (rRNA and MB antigen) genes, may reflect the phylogenetic relationship among organisms taxonomically classified as U. urealyticum.

DNA Primers↗
PubMed1989

A procedure for urease and protein extraction from staphylococci.

Staphylococcal cell protein and urease can be solubilized after growth in Todd-Hewitt broth supplemented with 0.5% yeast extract by extraction for 18-24 h in phosphate buffer, pH 7.0. In general 20% (but up to 100%) of the urease present in the original cells could be solubilized. Less protein was solubilized. Species examined included coagulase-negative staphylococci, Staphylococcus intermedius and Staph. aureus. Extracts of Staph. epidermidis prepared by this procedure gave electrophoretic urease and protein patterns similar to those prepared by sonication. The procedure was simple and minimized handling of the cells.

Bacterial Proteins↗
PubMed2004

Immunological rapid urease test using monoclonal antibody for Helicobacter pylori.

BACKGROUND AND AIM: The current diagnostic methods for detecting Helicobacter pylori infection include rapid urease test (RUT), urea breath test (UBT), histology, culture, and serum antibody detection. The present study evaluated the efficacy of a novel highly specific test, an immunological RUT (IRUT), that uses a monoclonal antibody against H. pylori urease. METHODS: The clinical evaluation of the IRUT was performed in 100 subjects. Each gastric mucus sample obtained during endoscopic examination was incubated for 15 min with a solid tip coated with monoclonal antibody for H. pylori urease, and then the tip was introduced into a pH-monitoring cell containing urea solution. The change in pH of the solution after the enzymatic reaction (delta pH) was measured. The performance of the IRUT was compared with culture, histology, RUT, and UBT. RESULTS: Of the 47 H. pylori-positive subjects, 43 were IRUT positive (sensitivity, 91.5%), and of the 53 H. pylori-negative subjects, 52 were negative (specificity, 98.1%). Compared with the usual diagnostic methods, IRUT had high sensitivity and specificity for the detection of H. pylori and was no less efficient. CONCLUSIONS: IRUT is a sensitive, specific and very rapid (within 20 min) method of detecting H. pylori infection.

Antibodies, Monoclonal↗
PubMed2006

Influence of urease activity in the intestinal tract on the results of 13C-urea breath test.

BACKGROUND AND AIM: A late rise in (13)CO2 excretion in the (13)C-urea breath test (UBT) should be found when the substrate passes rapidly through the stomach and makes contact with the colonic bacteria. The aim of this study was to evaluate the influence of intestinal urease activity on the results of the UBT. METHOD: A total of 143 subjects who were diagnosed as Helicobacter pylori negative by serology, histology and rapid urease test were recruited. At the end of endoscopy, the tip of the endoscope was placed to the second part of the duodenum and 20 mL of water containing 100 mg of (13)C-urea was sprayed into the duodenum. Breath samples were taken at baseline and at 5, 10, 20, 30 and 60 min after administration. RESULTS: Of 143 subjects, breath Delta(13)CO2 values higher than 2.5 per thousand were detected in six (4.2%), four (2.8%) and five (3.5%) subjects at 20, 30 and 60 min, respectively. There was no subject with high Delta(13)CO2 values at 5 and 10 min. Only one subject had an immediate rise at 60 min. CONCLUSION: Variability derived from urease activity in the intestinal tract appears to be minimal up to 60 min after ingestion of the test urea.

Adolescent↗
PubMed2000

Non-urease producing Helicobacter pylori in chronic gastritis.

BACKGROUND: Helicobacter pylori infection is the commonest cause of gastritis. Different patterns of immune response to H. pylori infection and characteristics of bacteria are considered to contribute to clinical outcomes. AIM: To determine characteristics of the host H. pylori relationship in subjects with non-ulcer dyspepsia and a histological diagnosis of gastritis. METHODS: Thirty-five subjects with chronic gastritis undergoing endoscopy (mean age 53 years, range 24-82, 14 male and 21 female) were studied, none of whom was on nonsteroidal anti-inflammatory drugs or antibiotics. H. pylori infection was determined by rapid urease test (CLOtest), culture, antibody and RT-PCR for Ure C, Cag A and 26 kDa gene and histology. Cytokine production of mucosal IL-6 and IL-8 were measured by ELISA. RESULTS: Fifteen subjects were positive by CLOtest and/or bacterial culture. In these subjects histology showed numerous helical forms of H. pylori (Group I). Nine subjects were negative by CLOtest, bacterial culture, and mRNA for urease C fragment, but positive by PCR for the 26 kDa protein encoding gene. Histology in these subjects showed the presence of either coccoid forms (four), or scant helical forms (two), or mixed coccoid/helical forms (three) (Group II). Eleven subjects were negative by all methods of detection (Group III). IgG and IgA antibody levels in serum (p<0.05) and gastric tissue culture supernatant (p<0.001) were significantly higher in Group I than those in Group II or III. There were significant differences in the IgG serum and IgA supernatant antibody levels (p<0.01 and p<0.05) when Group II was compared to Group III. Supernatant IL-6 levels were significantly higher in Group I (p<0.01) than those from Groups II and III. IL-8 levels were higher in Group I (p<0.01) and Group II (p<0.05) when compared to Group III. CONCLUSIONS: 'H. pylori-negative' gastritis can be associated with a non-urease producing form of H. pylori, with a reduction in both local and systemic antibody levels and mucosal pro-inflammatory cytokines.

Adult↗
PubMed1991

Relationship between urease-producing bacteria, urinary pH and encrustation on indwelling urinary catheters.

In 11 patients with long-term indwelling catheters the amount of catheter encrustation and urinary pH were measured and the urine regularly cultured over a prolonged period of time (median of 7 periods of 3 weeks). The mean urinary pH was related to the persistent presence of urease-producing micro-organisms (P. mirabilis) and urinary pH governed the precipitation of catheter encrustation. The critical pH appeared to be around 6.8. In patients with a mean urinary pH below this level the encrustation was minute (less than or equal to 2.9 mg phosphate). In patients with a mean urinary pH above 6.8 it was considerable but with a marked interindividual variation (35.5-138.7 mg phosphate). The composition of the encrustation was also strongly pH-related, with a much higher proportion present as magnesium ammonium phosphate in patients with a mean urinary pH above 6.8. The persistent presence of urease producers was not associated with a high pH or a more pronounced precipitation of phosphate in all patients. The amount of encrustation thus appears to depend not only on the presence of urease-producing micro-organisms but also on individual factors such as urinary composition.

Aged↗
PubMed2005

Effect of dietary anti-urease immunoglobulin Y on Helicobacter pylori infection in Mongolian gerbils.

BACKGROUND AND AIM: Helicobacter pylori is known to be a major pathogenic factor in the development of gastritis, peptic ulcer disease and gastric cancer. Recently, chicken egg yolk immunoglobulin Y (IgY) has been recognized as an inexpensive antibody source for passive immunization against gastrointestinal infections. The present study was designed to investigate the effect of anti-urease IgY on H. pylori infection in Mongolian gerbils. METHODS: H. pylori-infected Mongolian gerbils were administered a diet containing anti-urease IgY, with or without famotidine (F). After 10 weeks, bacterial culture and measurement of the gastric mucosal myeloperoxidase (MPO) activity were performed. In a second experiment, another group of gerbils was started on a diet containing F + IgY a week prior to H. pylori inoculation. After 9 weeks, these animals were examined. RESULTS: In the H. pylori-infected gerbils, there were no significant differences in the level of H. pylori colonization among the different dietary and control groups. However, the MPO activity was significantly decreased in the H. pylori group administered the F + IgY diet compared with that in the H. pylori group administered the IgY, F, or control diet. Furthermore, in the gerbils administered the F + IgY diet prior to the bacterial inoculation, inhibition of H. pylori colonization and suppression of the elevated gastric mucosal MPO activity were observed. CONCLUSIONS: Oral administration of urease-specific IgY not only inhibited H. pylori disease activity in H. pylori-infected gerbils, but also prevented H. pylori colonization in those not yet infected. These encouraging results may pave the way for a novel therapeutic and prophylactic approach in the management of H. pylori-associated gastroduodenal disease.

Animals↗
PubMed2006

Development and evaluation of a DNA vaccine based on Helicobacter pylori urease B: failure to prevent experimental infection in the mouse model.

BACKGROUND: The development of a vaccine against Helicobacter pylori has become a priority to prevent major morbidity and mortality associated with this infection. Our goal was to prepare and evaluate a DNA vaccine based on the urease B gene (ureB). METHODS: The ureB gene of H. pylori was amplified and cloned into the eukaryotic expression vector pcDNA3.1/TOPO. Plasmid DNA was purified from transformed Escherichia coli cells and used to immunize mice by the intragastric, intramuscular, intrarectal (40 micro g each) and intranasal (16 micro g) route, three doses every 2 weeks, with CpG oligodeoxynucleotide (ODN) as adjuvant. Four weeks after the third dose, animals were orally challenged with Helicobacter felis and were sacrificed 6 weeks later. The stomach was stained to detect the presence of infection. RESULTS: Despite in vitro confirmation of successful cloning and functionality of the ureB gene with expression of a protein morphologically and antigenically identical to urease B, the DNA vaccine did not perform well in vivo. Immunization of mice produced a weak immune response. Overall, intrarectal and intranasal administration seemed more immunogenic than other routes. Protection against challenge was modest and nonsignificant, and slightly better on animals immunized by the intramuscular and intranasal route. CONCLUSION: A DNA vaccine based on H. pylori urease B was poorly immunogenic and nonprotective at the conditions evaluated. Higher doses, better adjuvants or a prime-boost approach may circumvent these limitations.

Adjuvants, Immunologic↗
PubMed1994

Identification of mixed genotypes in Helicobacter pylori from gastric biopsy tissue by analysis of urease gene polymorphisms.

PCR-RFLP analysis of the urease A and B genes was used to investigate the frequency that Helicobacter pylori from gastric biopsy comprised genotypically distinct strains. Seventy-five strains of H. pylori from 44 subjects from four geographically diverse locations were examined and 31 distinct urease gene profiles were identified. For most cultures, the totals of the RFLP sizes were within 4% of the 2.41-kb PCR product. Five strains were genomically more complex suggesting they contained a mixture of related genotypes. The validity of the test confirmed by analysis of a known strain mixture and of single colony picks from biopsy cultures. Urease gene PCR-RFLP profiling was shown to be more sensitive than ribotyping for determining the genotypic homogeneity of H. pylori.

Base Sequence↗
PubMed2001

Genetic complementation of the urease-negative Helicobacter pylori mutant N6ureB::TnKm.

Helicobacter pylori produces urease composed of the structural subunits UreA and UreB. Isogenic mutants produced by shuttle mutagenesis from the wild-type strain N6 are widely used in the literature. We describe the genetic complementation of the mutant N6ureB::TnKm by stable transformation with the vector pHel2 containing the cloned genes ureA and ureB and their specific promoter sequence. The orientation of the cloned insert was found to be crucial for urease expression. The majority of complemented clones functionally expressed urease at higher levels than did N6. Homologous recombination between chromosomal and cloned genes occurred at a frequency of 5%.

Cloning, Molecular↗
PubMed1976

Localization of urease activity in Ureaplasma urealyticum cells.

Measurements of urease activity of various cell fractions of U. urealyticum showed that this activity was confined to the soluble fraction of the cytoplasm. It was attempted to devise a method for electron microscopic detection of the sites of urease activity based on precipitation of electron dense MnO2 at the alkaline pH created by hydrolysis of urea. The results obtained by this method supported the previous results indicating a cytoplasmatic localization of the urease activity in the cells. Helical ribosome patterns were observed when glutaraldehyde fixed cells were treated with the cytochemical test solutions.

Cell Membrane↗
PubMed1984

A urease-ELISA for the detection of mycoplasma infections in poultry.

An ELISA utilising a urease-antibody conjugate specific to chicken IgG was examined as an alternative to the serum agglutination and the haemagglutination inhibition tests in the diagnosis of Mycoplasma gallisepticum and M. synoviae infections in poultry. Use of a urease conjugate allowed the serum reactions to be appraised without the need for expensive photometric equipment. Non-specific binding of conjugate to antigen was eliminated by treatment of antigen coated microplates with 10% foetal calf serum in phosphate buffered saline. Some chicken serums produced non-specific reactions. These reactions were reduced without any loss of test sensitivity by making the initial 1:5 dilution of chicken serum in whole sheep serum rather than diluting buffer. Tests on serums from experimentally infected chickens showed that the urease ELISA was specific, and was as sensitive as the serum agglutination test but more sensitive than the haemagglutination inhibition test.

Animals↗
PubMed1976

Effect of organic mercurials and sulfhydryl compounds on the urease activity of Proteus: inhibition by urine and ascorbic acid.

Meralluride, mercaptomerin, ethacrynic acid, and penicillamine inhibited urease activity of Proteus mirabilis. The activity of the organic mercurials and ethacrynic acid was markedly inhibited by human and dog urine. Antiurease activity could not be detected in the urine of a human and a dog given meralluride by injection. Urine from patients receiving penicillamine also failed to inhibit urease activity. Ascorbic acid inhibited, whereas dehydroascorbic acid enhanced, the activity of the mercurials, but neither agent altered the inhibitory effect of urine. The lethal effect of meralluride against Proteus occurred at the same concentration at which urease activity was inhibited, but penicillamine inhibited the enzymatic activity without affecting viability of the organism. The data suggest that these sulfhydryl-reactive compounds will not be useful against Proteus infections of the urinary tract.

Animals↗
PubMed2000

Enhanced reduction of Helicobacter pylori load in precolonized mice treated with combined famotidine and urease-binding polysaccharides.

The present study investigated the effect of a model urease-binding polysaccharide in combination with a histamine H(2) receptor antagonist on Helicobacter pylori colonization in vivo. Euthymic hairless mice were treated daily with dextran sulfate via drinking water and/or famotidine via intragastric gavage starting at 1 week postchallenge with a CagA(+) VacA(+) (type 1) strain of H. pylori. Treatment of precolonized mice for 2 weeks with dextran sulfate combined with famotidine yielded a group mean bacterial load (per 100 mg of gastric tissue) of log(10) 1.04 CFU, which was significantly lower than those of the famotidine (log(10) 3.35 CFU, P < 0.01) and dextran sulfate (log(10) 2.45 CFU, P < 0.05) monotherapy groups and the infected nontreated group (log(10) 3.64 CFU, P < 0.01). Eradication was achieved after 2 weeks of treatment in 50% or more of the test mice using drug combinations (1 or 2 weeks of famotidine plus 2 weeks of dextran sulfate) versus none in the monotherapy and positive control groups. The enhanced activity of the drug combination may be related to the daily pattern of transient acid suppression by famotidine inducing periodic bacterial convergence to superficial mucus sites penetrated by dextran sulfate from the lumen. Increased urease-dextran sulfate avidity was observed in vitro in the presence of famotidine and may partly account for the enhanced activity. With potential utility in abbreviating treatment time and eradication of antibiotic-resistant strains, the use of urease-targeted polysaccharides concurrently with a gastric acid inhibitor warrants consideration as an additional component of the standard multidrug chemotherapy of H. pylori infection.

Animals↗
PubMed1969

Four-hour urease test for distinguishing between Klebsiella and Enterobacter.

Infections with Klebsiella and Enterobacter have increased among hospitalized patients. To study such infections, relatively simple but precise methods are needed for clinical laboratories to identify the two genera accurately. Moreover, a rapid identification is essential for assisting with the therapy of the patients. For this purpose, a new 4-hr urease test was developed so that colonies could be tested directly from blood-agar plates which have been inoculated with clinical material and allowed to incubate overnight. This 4-hr test was positive with 98.5% of 202 Klebsiella species and negative with 80 Enterobacter species. As a single criterion for distinguishing between the two major genera, the new 4-hr urease test was just as accurate as a motility test (99% of the 282 isolates were accurately identified with either). The 4-hr urease test represents a simple, rapid, and reliable technique which is ideally suited for use in clinical microbiology laboratories.

Bacteriological Techniques↗
PubMed1990

Helicobacter pylori urease activity is toxic to human gastric epithelial cells.

A human gastric adenocarcinoma cell line was used to evaluate the contribution of urease from Helicobacter (formerly Campylobacter) pylori to its cytotoxicity. Gastric cells cultured in medium supplemented with 20 mM urea were exposed to 5 x 10(6) CFU of H. pylori per ml with or without the addition of a urease inhibitor, acetohydroxamic acid. Viabilities of cells exposed to H. pylori for 2, 24, and 48 h, assessed by incorporation of neutral red dye, were 60, 27, and 16%, respectively; however, the viabilities of cells exposed to both H. pylori and acetohydroxamic acid were 92, 46, and 20% after 2, 24, and 48 h, respectively, (P less than 0.001). Therefore, the urease activity of H. pylori may play an important role in its pathogenicity, and inhibition of this enzyme activity may have therapeutic potential.

Ammonia↗
PubMed1991

Isolation and detection of urease genes in Ureaplasma urealyticum.

Urease from ureaplasmas was purified by immunoaffinity chromatography, and the N-terminal amino acid sequence was determined for two of the three subunits. These sequences were used to design primers for a polymerase chain reaction (PCR) that amplified most of the gene coding for one of the subunits. By using a novel "PCR walking" technique, we synthesized almost the complete locus on two overlapping PCR products. We present here a partial nucleotide sequence of the urease locus from Ureaplasma urealyticum (serotype 8), which agrees with our N-terminal amino acid data but differs slightly from the sequence previously reported (A. Blanchard, Mol. Microbiol. 4:669-676, 1990). Also described are PCR primers, intended for diagnostic use, that amplify a sequence from all Ureaplasma strains tested but not from any other mycoplasmas or urease-positive bacteria.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of Helicobacter mustelae urease.

Helicobacter mustelae is a urease-rich bacterium associated with gastritis in ferrets. The ureases of H. mustelae and Helicobacter pylori, a bacterium implicated in human gastritis, share many characteristics. Helicobacter sp. ureases appear to be unique among bacterial enzymes in exhibiting submillimolar Km values and in being composed of two subunits.

Campylobacter↗