Urease Assay Research Articles

Expression of UreI is required for intragastric transit and colonization of gerbil gastric mucosa by Helicobacter pylori

Helicobacter pylori colonizes the antral mucosa of the human stomach. There is a controversy as to whether the microorganism is exposed to acidity in its ecological niche. In vitro, the microorganism requires urease for gastric colonization and survival at pH < 4.0. UreI encodes an acid activated urea channel enabling urea access to intrabacterial urease at acidic pH. UreI is also necessary for survival at pH < 4.0. However, the role of UreI for both intragastric transit and colonization of the epithelial gastric mucosa has never been analyzed in detail. We therefore infected gerbils, whose intragastric pH and response to infection resemble those of man, with H. pylori G1.1 wild type bacteria and their corresponding isogenic ureI mutants. Inhibitors of gastric acid secretion and colonization were used for manipulation of gastric pH. Gastric colonization was determined by urease assay and PCR. Gastric pH was measured with pH electrodes. Whereas H. pylori wild type or ureI complemented ureI knockout bacteria colonized the antrum, ureI deletion mutants were unable to colonize. However, continuous inhibition of acid secretion resulted in gastric colonization by the ureI mutants, as also observed with the wild type strain. Restoration of acid secretion resulted in eradication of ureI mutants but not wild type bacteria. The data show that ureI is essential for both gastric transit after inoculation and mucosal colonization in the untreated stomach. The eradication of ureI mutants following restoration of acid secretion suggests that the organism is exposed to pH < 4.0 at the surface of the antral mucosa and that UreI provides a target for specific monotherapy of H. pylori infections.

Further development of the Helicobacter pylori mouse vaccination model

Immunisation against Helicobacter infection in mouse models has thus far produced neither complete protection against the bacteria, nor a complete prevention of the associated gastritis. This study aimed firstly to compare the sensitivities of the various methods used to assess H. pylori infection in the mouse model, and secondly to develop the experimental design to induce a more effective immunity, aimed at further reducing bacterial burden in the gastric tissue. Various mouse strains were prophylactically immunised with whole bacterial sonicate and cholera toxin before challenge with H. pylori-SS1. The relative sensitivities of the urease assay, histological assessment and the colony forming assay to detect levels of H. pylori colonisation were compared. Comparisons of different antigen doses and different timecourses of immunisation were performed. The colony forming assay was found to be far more sensitive than either the urease assay or histological assessment for determining the protective efficacies of immunisation. Mice which had 10 5 H. pylori per gram of stomach by colony assay were negative by histology and urease. Lower doses of whole cell sonicate were more protective than high doses and more effective immunisation was achieved by leaving at least 3 weeks between immunisation instead of weekly immunisations. In conclusion, for assessment of H. pylori colonisation in the mouse model, the colony forming assay should be used. The experimental protocol for immunisation has been altered to produce a significant improvement in protection. However, full protection has still not yet been achieved and more work is still required.

Oral immunization with recombinant Helicobacter pylori urease confers long-lasting immunity against Helicobacter felis infection

Recombinant Helicobacter pylori urease (rUre) has been shown to confer protection against challenge with Helicobacter felis in mice. The purpose of the present study was to examine duration of the immune response and long-term protective efficacy of immunization with rUre. Swiss Webster mice were orally immunized four times at weekly intervals with 100 μg rUre plus 5 μg heat-labile enterotoxin of Escherichia coli (LT) adjuvant, or with LT only. At 4, 10, 20 or 40 weeks post immunization, 25 rUre-immunized mice and 25 control mice were challenged with H. felis and sacrificed at 2 or 10 weeks post-challenge. H. felis infection was assessed by gastric urease assay and by histology. Anti- H. pylori urease specific antibody levels were measured in serum and saliva both pre- and post-challenge. Over the 40 week time period, the infection rates in rUre-immunized mice were significantly lower than those in controls ( p<0.05) as assessed by gastric urease activity. Protection ranged from 79–100% at 2 weeks post-challenge and 63–78% at 10 weeks post-challenge. Gastric bacterial density in rUre-immunized mice was significantly lower than that of controls ( p<0.03) as determined by histologic assessment. Anti-urease antibody levels remained elevated in the serum and mucosal compartments at 39 weeks following immunization. This study shows that immunization with rUre plus LT results in long-lasting protective immunity against challenge with H. felis.

A simplified urea breath test for the diagnosis of Helicobacter pylori infection using the LARA System. Laser Assisted Ratio Analyzer.

Helicobacter pylori, one of the most prevalent human pathogens, is associated with chronic gastritis, peptic ulcer disease, and possibly gastric cancer and primary gastric lymphoma. The need to treat these patients has necessitated the development of improved methods to diagnose H. pylori infection. We present the preliminary assessment of a 13C-urea breath test (UBT) in which the expired 13CO2 is detected in a rapid, simple, inexpensive way by the LARA (Laser Assisted Ratio Analyzer) System (Alimenterics, Inc., Morris Plains, NJ). Eighty-seven consecutive patients, examined for upper gastrointestinal symptoms, underwent endoscopy. H. pylori infection was established by antral biopsies and a rapid urease test (CLOtest). The UBT was performed between 2 and 24 hours after endoscopy. Of the 84 analyzable patients, 70 were found to be H. pylori-positive either by histology or by CLOtest. All 70 were positive by the LARA UBT, yielding a sensitivity of 100%. Fourteen patients were negative for H. pylori by histology and the CLOtest. Of these, 12 were negative by the LARA UBT and 2 were positive, yielding a specificity of 85.7%; because of the limitations of H. pylori detection by histology or urease assays, however, the specificity of the UBT may have been underestimated. Our study demonstrates the feasibility of a nonradioactive, rapid UBT based on the LARA system and suggests the need for its more detailed evaluation.

Apoptosis as a marker for the gastric epithelial cell damage caused by Helicobacter pylori infection

We investigated the relationship between apoptosis and gastric epithelial ceils infected with Helicobacter pylori (H. pylori). Patients: Patients examined with included 24 with gastric ulcer(GU), 30 with duodenal ulcer(DU), and 7 with gastric and duodenal ulcer(GDU). The diagnosis was endoscopically determined, and H. pylori infection was confirmed either by bacterial culture or by histological examination. The control group consisted of 5 healthy volunteers. Methods: At endoscpical examination, six biopsy specimens were obtained from the stomach. Three were used for the apoptosis study, (the epithelial cells were fixed with 75% ethanol stained with Propidium Iodine and apoptotic ceils were determined by flowcytometry). The other three were used for a bacterial culture (H. pylori No. VTassay, Urease assay) and histological examination(gastritis score). Results: The percentages of apoptotic in the gastric epithelial ceils in GU, DU, GDU and healthy volunteers were 21.2_2.1%, 21.4±2.3%, 17.0_3.1%, and 7.4-+2.0% respectively. Statistical significance was noted between the ceils from the total H. pylori -positive patient group and those from the healthy volunteers (p < 0.05). Interestingly, the percentage of apoptosis in the cells from the DU patients in the active stage was significantly higher than those from DU patients in healing and scar stages. (30.3 -+ 4.8% vs 19.7 -+ 4.2% 16.9 -+ 2.3 p < 0.01). The percentage of apoptotic cells did not differ significantly between patients with different histological score, No. of H. pylori and VTassay. The percentage of apoptotic cells was correlated with urease activity. Conclusion: Apoptosis in the gastric epithelial cells is correlated not only with H. pylori infection, but also with the stage of peptic ulcer. Thus, apoptosis may be a useful marker for the mucosal damage evoked by 1-1. pylori infection.

Diagnosis and Treatment of Helicobacter pylori Infection Among California Medicare Patients

Antibiotic treatment of Helicobacter pylori infection in active peptic ulcer disease has been demonstrated to speed ulcer healing, reduce the risk of rebleeding, and prevent long-term recurrence. The objective of this study was to determine whether Medicare patients with peptic ulcer disease who are admitted to acute care hospitals are being tested or treated for H pylori infection as recommended by a National Institutes of Health consensus panel. The study was designed as a retrospective medical records survey. From the Medicare National Claims History File, all persons 65 years and older admitted to California fee-for-service hospitals for peptic ulcer disease in 1994 were identified. A random sample of 600 claims was selected for review. After exclusions, 524 patients were eligible for study. The main outcome measures were (1) the proportion of patients who were tested for H pylori infection by 1 of the 5 available methods (histopathologic study, urease assay, microbiologic culture, serum antibody testing, or urea breath test) and (2) the proportion who were treated with antibiotics (amoxicillin, tetracycline, clarithromycin, or metronidazole) for H pylori infection. Thirty-nine percent of patients with peptic ulcer disease were tested for H pylori infection and 3% were treated empirically. Only 47% of the patients who had a positive diagnostic test result for H pylori were treated with antibiotics. Because diagnosis and treatment of H pylori infection has been demonstrated to improve outcomes and decrease expenses, the data indicate a substantial opportunity to improve the care of elderly Medicare patients with peptic ulcer disease.

Characterization of feline Helicobacter pylori strains and associated gastritis in a colony of domestic cats.

Twenty-four young adult domestic cats from a commercial vendor were found to be infected with Helicobacter pylori. Histopathologic analyses, selected electron microscopy, and urease mapping were performed on mucosal samples collected from the cardias and fundi, bodies, and antra of these cats' stomachs. H. pylori organisms were abundant in all areas of the stomach on the basis of histologic evaluation and urease mapping. H. pylori infection was associated with a moderate to severe lymphofollicular gastritis in 21 of 24 cats (88%). The gastritis was most pronounced in the antral region and consisted mainly of multifocal lymphoplasmacytic follicular infiltrates in the deep mucosa. The severity of gastritis in the antrum corresponded to high numbers of H. pylori there on the basis of the use of the urease assay as an indicator of H. pylori colonization. Ten of 24 cats (42%) also had small to moderate numbers of eosinophils in the gastric mucosa. All 24 cats had gastric lymphoid follicles, with follicles being most prevalent in the antrum. Electron microscopy of gastric tissue revealed numerous H. pylori organisms, some of which were closely adhered to the mucosal epithelium. Human H. pylori gene-specific primers to ureA and ureB amplified products of similar sizes from H. pylori cat isolates. Digestion of the products with restriction enzymes resulted in fragments characteristic of the restriction fragment length polymorphism patterns of H. pylori isolates from humans. In the domestic cat, H. pylori infection is associated with a lymphofollicular gastritis, consisting of lymphocytic and plasmacytic infiltration into the lamina propria, and the organism appears to provide chronic antigenic stimulation resulting in the formation of gastric lymphoid follicles.

Assessment of urease inhibition assays for measuring toxicity of environmental samples

We evaluated the potential of free and immobilized urease for assaying toxicity of environmental samples. Free urease was sensitive to heavy metals and insensitive to organic toxicants. Toxicity of industrial wastewaters and process water, as determined via free urease assay, was correlated with the heavy metal content of these samples. High background (blank) absorbance was encountered in samples containing arnmonia. The background exceeded the linear range of the assay at ammonia nitrogen concentrations exceeding 8 mg/l. Immobilizing urease on glass beads was considered as a means of overcoming the problem of ammonia interference. Immobilized enzyme was unfortunately not sensitive to heavy metals. The potential of free urease for toxicity testing cannot be fully realized until a means of overcoming ammonia interference is developed.

Topographic Variation of Soil Nitrogen Dynamics at Walker Branch Watershed, Tennessee

Abstract Understanding the spatial and temporal variability of soil nitrogen (N) transformations is central to quantifying the N dynamics and productivity of ecosystems. The objectives of this work were to examine spatial and temporal variation of soil N dynamics and to identify factors correlated with topographic variation in soil N dynamics in a forest watershed. Net N mineralization and net nitrification potential were measured by aerobic laboratory incubations of surface (0-7 cm) mineral soils. Principal components analysis was used to describe sampling sites across the watershed based on 13 site characterization variables. A topographic index used in hydrologic modeling, In (α/tan β), was calculated for each site as the natural logarithm of the ratio of the upslope drainage area per unit contour length (α) to the local slope angle (tan β). Soils from valley floors had greater total N concentrations, lower carbon-to-nitrogen (C:N) ratios, greater potential net nitrification, and greater microbial activity (as indicated by short-term urease assays) than soils from ridges. Mean net nitrification potential was 0.59 μg N g-1 d-1 in surface soils from valley floors and was &amp;lt; 0.01 on ridges and slopes. The first principal component was related to the N and C properties of soils, leaf litter, and leaf fall at a site. The second principal component was related to forest stand composition. The topographic index was significantly correlated with important variables related to soil N dynamics. Once calibration data are derived, this index may be useful as a first approximation to total soil N concentrations and soil C:N ratios in forest watersheds because In (α/tan β) can be calculated from geographic information systems that contain topographic data. For. Sci. 40(3):497-512.

Animal and public health implications of gastric colonization of cats by Helicobacter-like organisms.

The bacterial genus Helicobacter contains a number of species which colonize the gastric mucosa of mammals. Natural and/or experimental gastric pathology has been correlated with colonization in humans and a wide variety of animal species. Historical reports in the literature suggest that a high percentage of cats are colonized by large, spiral, gastric helicobacter-like organisms (GHLOs). One of these bacteria (Helicobacter felis) has been isolated on artificial media and has experimentally caused gastritis in gnotobiotic dogs. This study surveyed the prevalence of helicobacter colonization in random-source cats by using the urease assay. Histologic examination was performed to determine the degree of associated pathology present. GHLOs associated with chronic gastritis were present in 70% of the juvenile and 97% of the adult cats studied. Although further study is needed to determine specifically what role GHLOs play in feline gastrointestinal disease, these results indicate that helicobacter colonization should be considered in the pathogenesis of feline gastroenteropathy. Furthermore, the high prevalence of feline infection is interesting because cats have recently been implicated as a potential reservoir for human infection by helicobacter-like organisms.

Gas phase derivatization of ammonia with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole and its application to urease assay.

An ammonia-specific and rapid fluorometric method for determination of ammonia and urease activity was developed. The method is designed to assay ammonia levels or urease activity for the rapid diagnosis of Helicobacter pylori infection. 4-Fluoro-7-nitrobenzo-2-oxa-1,3-diazole was used to derivatize ammonia and 4-amino-7-nitrobenzo-2-oxa-1,3-diazole was analysed by high performance liquid chromatography at an excitation wavelength of 455 nm and an emission wavelength of 520 nm. Derivatization was designed to react with ammonia gas produced in a strong alkaline pH sample. The fluorescent intensity was linear in the range of 0.1-10 mM ammonia per tube when the reaction was carried out for 15 min at 37 degrees C. Urease activity, judged as the amount of ammonia production from urea, could be measured at 25 ng per tube (S/N = 1.5) with Jack bean meal urease. Because of its rapidity, this assay is potentially superior to the current standard method in use in clinical settings.

Helicobacter felis gastritis in gnotobiotic rats: an animal model of Helicobacter pylori gastritis

The gastric spirillum Helicobacter felis, originally isolated from the cat stomach, colonizes the stomachs of germfree rats. Studies were designed to examine the pathological and serological responses of germfree rats inoculated orally with H. felis. At 2 weeks postinoculation, the gastric mucosa of germfree rats had lymphocytes and eosinophils scattered in small foci throughout the subglandular region of the antrum. Small numbers of lymphocytes were present in the subglandular portion of the antral mucosa that focally extended through the lamina propria towards the luminal surface. Eight weeks postinoculation, the inflammation was confined to the antrum. It was characterized by increased numbers of lymphocytes and eosinophils in the subglandular areas, with focal aggregates of lymphocytes in the submucosa. Some lymphoid aggregates extended from the submucosa through the muscularis mucosa and lamina propria to the luminal surface. H. felis was demonstrated with the Warthin-Starry stain, bacterial culture, and urease assay, particularly in the antrum. H. felis also produced a significant immunoglobulin G antibody titer at 2, 4, and 8 weeks postinoculation as well as a transitory immunoglobulin M response at 2 to 4 weeks postinoculation. Contact control rats were not infected, inferring that fecal-oral spread of H. felis did not occur.

Urease inhibition by EDTA in the two varieties of Cryptococcus neoformans.

Cryptococcus neoformans var. neoformans (74 isolates) and C. neoformans var. gattii (44 isolates) were used to test urease activity after growth on both yeast extract-glucose-peptone agar (YEPG) and on YEPG supplemented with 100 microM EDTA. Every isolate grown on YEPG agar for 48 h at 30 degrees C produced a positive reaction within 1 h in a modified rapid urease assay at 37 degrees C. However, isolates grown on YEPG with 100 microM EDTA showed a distinct pattern which corresponded to their varietal status. All but 1 of 74 C. neoformans var. neoformans isolates (98.7%) produced a positive reaction within 1 to 4 h, while none of 44 C. neoformans var. gattii isolates produced a positive reaction within the same period. The urease inhibition results and the canavanine-glycine-bromthymol blue agar test results showed 100% correlation among isolates of C. neoformans var. gattii and 98.7% correlation among isolates of C. neoformans var. neoformans. Two representative isolates of C. neoformans var. gattii (serotypes B and C) were further tested for urease during a prolonged incubation period in urea broth. These isolates failed to show a positive reaction even after 11 h of incubation. The uptake of EDTA was negligible in the two varieties. Extracts of cells grown on YEPA agar showed a high level of urease activity in both varieties. Extracts of cells grown on the agar with 100 microM EDTA showed a marked reduction (86%) of urease activity in one isolate of C. neoformans var. gattii but showed only a 30% reduction in one isolate of C. neoformans var. neoformans. Based on these results, the differential effect of EDTA on the two varieties of C. neoformans appeared to be due to greater inhibition of urease synthesis in C. neoformans var. gattii.

Comparison of methods for the analysis of dissolved urea in seawater

A comparison between the diacetyl monoxime and urease methods for measuring dissolved concentrations of urea in seawater was conducted in artificial seawater, phytoplankton-culture filtrate and both natural and ureaspiked field samples from coastal and oceanic enviroments during 1984. The urease technique underestimated urea concentrations in unbuffered photoplankton-culture filtrate as a result of the inhibition of the urease enzyme, causing the incomplete hydrolysis of urea in these samples. Factors responsible for inhibiting urease included pH, seawater ions, and possibly extracellular metabolites produced in unialgal cultures. Seawater type and time of sample collection were important variables affecting urea measurement by the urease method, and recovery of internal standards ranged from 40 to 100%. Increasing the heating time of the urease assay, or the concentration of urease added to the seawater samples increased the amount of urea determined by the urease method. However, measured values were still less than the concentration of the urea internal standards. The diacetyl monoxime method was suitable for urea determinations in all the seawater samples we examined; it was easily automated, and the results were accurate and reproducible. This modified technique is recommended for measuring disolved concentrations of urea in seawater.

Effect of assay conditions and field exposure on urease activity associated with cereal residues

Abstract Urea fertilisers are often applied to soil with cereal residues on the surface. Although the urease properties of soils have been investigated, the urease activities associated with cereal residues have not been thoroughly studied. Two experiments were performed to develop an acceptable urease assay for cereal residues, and to determine the effect of field exposure and concomitant saprophytic colonization on urease levels. Two acceptable assays were found: 1) incubation in the presence of THAM‐H2SO4 buffer (pH 8), toluene, and urea, and 2) incubation only in the presence of urea. In both assays substrate concentration was 0.05 M, incubation temperature was 37°C, incubation length was 2 hours, and urea hydrolysis was determined by ammonium production. Although both assays were highly correlated (r2 = 0.992), the buffer method gave higher values. Subsequent investigations illustrated that urease activity associated with cereal residues is dramatically increased by saprophytic colonization after har...

Urease assay and ammonia release from leaf tissues

The rate of release of ammonia from leaf tissues of Glycine max and Triticum aestivum suspended in urea solutions was relatively low for initial periods of up to 40 min, due to retention of ammonia by the tissues. Urease, EC 3.5.1.5, could be assayed by the subsequent rate of ammonia release. The method was applied to leaves of 29 other species.

The effects of substrate flow-rate on immobilized urease assays

Urease (urea aminohydrolase, EC 3.5.1.5) has been immobilized on o-alkylated nylon tubes in an enzymatically active form. The measured activity of the enzyme coil has been shown to be dependent on the flow rate of substrate through it. The apparent K m decreased and V increased as flow rates were increased. It is suggested that diffusional control is responsible for these observations. Their implications in flow-through immobilized enzyme analyses are discussed.

Urease assay and urease-producing species of anaerobes in the bovine rumen and human feces.

A growth medium and test were developed for rapid detection of urease in fermentative anaerobic bacteria. Using nonselective rumen fluid roll-tube agar medium and the new test, it was confirmed that Peptostreptococcus productus is often the most numerous urease-forming species in human feces. Also, some fecal strains of Ruminococcus albus, Clostridium innocuum, and Clostridium beijerinckii produced urease. Single strains of Fusobacterium prausnitzii, Coprococcus catus, and Streptococcus mitis that were strongly ureolytic on isolation later lost this ability. Urease activity was also detected in many strains of nonselectively isolated rumen species. They include Succinivibrio dextrinosolvens, Treponema sp., Ruminococcus bromii (not previously known to be present in the rumen), Butyrivibrio sp., Bifidobacterium sp., Bacteroides ruminicola, and P. productus. Most P. productus strains contain urease; however, the uniformity of this feature in the other species noted above is not known. The urease in many of these species was not detected if the growth medium contained 0.2% or more (each) yeast extract and Trypticase.

Effect of Jackbean Urease Injections and Chlortetracycline on Rate of Gain and Feed Efficiency in Swine

Two trials involving fifty-five Yorkshire × Poland China barrows were conducted to study the effects of immunity to jackbean urease on rate of gain and feed efficiency in swine. In the first trial, with factorially-arranged treatments including 100 ppm chlortetracycline in the diet and 100 Sumner units of urease injected intraperitoneally, chlortetracycline improved (P<.01) rate of gain and feed efficiency while the urease did not. A high initial serum titer against urease was observed, and further injections failed to increase this titer. In the second trial injections of up to 300 Sumner units of urease failed to increase the initial titer against urease and had no effect on feed efficiency and rate of gain. A technique of assaying for antiurease titer that involves a direct urease assay is reported.