Influence of pH on metabolism and urease activity of Helicobacter pylori

BACKGROUND & AIMS: The metabolic and urease responses of Helicobacter pylori to variations in gastric acidity are unknown. The aim of this study was to determine effects of changes of environmental pH on metabolism, urease activity, and survival of H. pylori in an unbuffered environment. METHODS: Bacterial metabolism and urease activity were determined by measuring pH changes in perfused microphysiometer chambers over a pH range from 2.5 to 9.0 with or without urea and survival by restoration of metabolism at pH 7.4. RESULTS: Glucose metabolism by acid-adapted H. pylori occurred at a perfusion pH between 3.5 and 8.6 and was highest between 7.4 and 8.2. Metabolism was irreversibly inhibited at pH <3.5 or >8.6. In the presence of 2.5 mmol/L urea, the chamber pH increased to about 6.2 during perfusion between pH 5.5 and 4.0. At pH 4.0 and below, urease activity increased several-fold without change of chamber pH. Urea in the perfusate enabled retention of metabolism after acid exposure but was toxic at pH 7.4. CONCLUSIONS: The metabolic range of acid-adapted H. pylori is between an environmental pH of 3.5 and 8.6. Extracellular pH-regulated internal urease activity allows metabolism in the pH range between 4.0 and 2. 5 by maintaining periplasmic pH at 6.2. The organism is an acid-tolerant neutralophile due to internal urease activity.     

Inhibition of Helicobacter pylori urease activity by ebrotidine

Lipopolysaccharide (LPS), known as one of the potent activators of macrophages, has inhibitory effects on the proliferation of normal macrophages and macrophage-like cell lines. We report here that LPS dose- and time-dependently suppressed the tritiated thymidine ([3H]TdR) incorporation into the acid-insoluble fraction with a significant inverse correlation to the tumour necrosis factor-alpha (TNF) production in the J774.1 macrophage cell line. Among the three tested enzymes involved in DNA synthesis, only thymidine kinase (TK) activity decreased progressively in parallel with the decline in [3H]TdR incorporation, reaching 97% inhibition within 12 hr of LPS treatment, while changes in the activities of other two enzymes, DNA polymerase alpha and thymidylate synthase (TS), were less significant. On the other hand, LPS inhibited the cell proliferation only incompletely, as judged by 62% inhibition of cell growth at 36 hr. Even in the experiments done in a TdR-free medium, cell growth was inhibited by LPS to the same extent, suggesting that TK was not directly involved in the proliferation of J774 cells. LPS also inhibited the conversion of TdR to thymidine monophosphate (TMP) in murine peritoneal exudate macrophages (PEM). Thus LPS-induced suppression of TdR salvage related to TNF production is common in both normal and neoplastic macrophages, and therefore may be of potential importance in the process of macrophage activation.     

In vitro activity of omeprazole in combination with several antimicrobial agents against clinical isolates of Helicobacter pylori

An agar dilution checkerboard method was used to evaluate the in vitro activity of omeprazole combined with clarithromycin, amoxicillin, and ceftibuten, respectively, against clinical isolates of Helicobacter pylori. Mueller-Hinton agar plus 5% horse blood, an inoculum of 10(6) cfu/ml, and incubation of 72 h in a CO2 atmosphere were used. With the omeprazole and clarithromycin combination, synergism was observed in 51.5% and partial synergism in 39.3% of 33 strains; with omeprazole and amoxicillin, synergism was observed in 3% and partial synergism in 60.6% of 33 strains; and with omeprazole and ceftibuten, synergism was observed in 33.3% and partial synergism in 50% of 24 strains. Antagonism between omeprazole and each antibiotic was exhibited in 0%, 6.1%, and 4.1% of these groups of strains, respectively. Of the antibiotic combinations tested, omeprazole plus clarithromycin exhibited synergism (partial or total) in the highest percentage of strains.     

Allelic exchange mutagenesis of nixA in Helicobacter pylori results in reduced nickel transport and urease activity

Helicobacter pylori, an etiologic agent of gastritis and peptic ulceration in humans, synthesizes urease, a nickel metalloenzyme, as its most abundant protein. NixA, a high-affinity nickel transport protein, allows synthesis of catalytically active urease when coexpressed with H. pylori urease in an Escherichia coli host. To determine whether NixA is essential for the production of active urease in H. pylori, nixA was insertionally inactivated with a kanamycin resistance cassette (aphA) and this construct was electroporated into H. pylori ATCC 43504; allelic exchange mutants were selected on kanamycin-containing medium. The nixA mutation, confirmed by PCR, reduced urease activity by 42% (140 +/- 70 micromol of NH3/min/mg of protein in the mutant versus 240 +/- 100 micromol of NH3/min/mg of protein in the parent (P = 0.037). Rates of nickel transport were dramatically reduced (P = 0.0002) in the nixA mutant (9.3 +/- 3.7 pmol of Ni2+/min/10(8) bacteria) of H. pylori as compared with the parent strain (30.2 +/- 8.1 pmol of Ni2+/min/10(8) bacteria). We conclude that NixA is an important mediator of nickel transport in H. pylori. That residual nickel transport and urease activity remain in the nixA mutant, however, provides evidence for the presence of a redundant transport system in this species.     

Novel bismuth compounds have in vitro activity against Helicobacter pylori

We examined the in vivo effects of ONO-5046xNa, a specific neutrophil elastase (NE) inhibitor, on the growth of 2 non-small cell lung cancer cell lines, EBC-1 and PC-3, transplanted into severe combined immunodeficiency (scid) mice. The daily intraperitoneal injection of ONO-5046xNa (50 mg/kg/day) completely suppressed the tumor growth in EBC-1, a human squamous carcinoma cell line which produces immunoreactive NE. By contrast, in PC-3, a human adenocarcinoma cell line which is unable to produce immunoreactive NE, the ONO-5046xNa treatment caused delayed growth of the tumor. These findings suggest that ONO-5046xNa may have a clinical role in preventing the growth of human non-small cell lung cancer.     

Systemic immunization with urease protects mice against Helicobacter pylori infection

The ability of systemic immunization to induce protection against Helicobacter pylori infection has been evaluated in a mouse model. It was observed that if appropriate formulations and adjuvants were used such immunization elicited in outbred Swiss mice levels of protection similar or better than those induced by the oral route in the presence of cholera toxin or Escherichia coli heat labile toxin. Recombinant urease mixed with adjuvants, which induced strong Th1 and Th2 responses elicited better protection than urease mixed with adjuvants which induced a predominant Th2 type response only. These experiments demonstrate the feasibility of parenteral immunization against H. pylori and suggest that an appropriate balance between Th1 and Th2 type responses is required to achieve complete protection.     

Cytotoxic effect of ammonia produced by Helicobacter pylori urease on the cultural cells

We investigated the action of the ammonia produced by Helicobacter pylori urease on the cultured cells. The urease was purified from supernatant fluid of sonicated cell of H. pylori cultured on blood agar for 2 days at 37 degrees C under microaerophilic condition. Purification was carried out by DEAE-Sepharose chromatography, Phenyl-Sepharose chromatography, Sephacryl S-200 SF chromatography and fast protein liquid chromatography on Mono-Q. Vero, HeLa and Intestin 407 cells with or without the addition of 30 mM urea were exposed to the purified urease. Those cells showed cytotoxic effects within 80 minutes after addition of purified urease in the presence of urea. The ammonia production was observed on tissue culture medium within 10 minutes, and the ammonia concentration ranged from 5.56 mg/ml to 7.3 mg/ml and pH in the medium was over pH 9.0. No such effect was observed on the cells exposed to urease without urea. Ammonia water added to Vero cells showed the same cytotoxic effect within 70 minutes on the production of ammonia and raised the pH. However, when the cells were exposed to the ammonia water pre-neutralized to a given pH 7-8 using 1 N HCl cytotoxic effect was not observed. It was concluded that the cytotoxic effect of H. pylori urease was dependent on ammonia generated by hydrolysis of urea.     

Culture of Helicobacter pylori from gastric biopsies transported in biopsy urease test tubes

Gastric biopsy specimens of 57 consecutively observed dyspeptic patients were studied for the presence of Helicobacter pylori by histological examination, biopsy urease test (BUT) and culture. For culture, biopsy samples were transported in both Stuart media and BUT tubes. All 15 isolates could be cultured from both Stuart and BUT tubes. Thus, if the main reason for culture of Helicobacter pylori is for antimicrobial susceptibility testing, only positive BUT tubes need to be submitted. This would reduce both the expense and the number of biopsies needed.     

Comparison of two rapid urease tests for detection of Helicobacter pylori infection

The selection of NIH 3T3 cells expressing a hydroxytamoxifen-inducible c-Raf-1-estrogen receptor fusion protein (c-Raf-1-BxB-ER) in the absence or presence of the inducer results in dramatic differences in the expression levels of the fusion protein. Hydroxytamoxifen-mediated constitutive activation of the Raf signal favors the selection of cells expressing low levels of c-Raf-1-BxB-ER. Cells selected in the absence of hydroxytamoxifen express up to 20 times higher levels of the inducible Raf kinase. Activation of the oncogenic Raf kinase in cells expressing low levels leads to a weak activation of the Raf/Mek/Erk cascade and the induction of S phase in confluent cells. The activation of cells expressing high levels of the kinase leads to a strong persistent signal and inhibits DNA synthesis and mitosis in proliferating cells. The inhibition of DNA synthesis and cell division is presumably due to the elevated expression of the cyclin-dependent kinase inhibitor p21cip1, similar to cells exposed to ionizing radiation. Despite the inhibition of DNA synthesis and mitosis, the constitutive activity of the Raf signaling pathway is still able to initiate cell growth. Activation of the high-intensity Raf signal in arrested serum-starved cells induces cell growth up to a size corresponding to that of M-phase cells in the absence of DNA synthesis. High-intensity Raf signals in proliferating cells consistently lead to an accumulation of cells with the size of M-phase cells and the DNA content of G1 cells or G2-M-phase cells. Therefore, the activation of Raf kinase is sufficient to drive cell growth, even in the presence of high levels of the cyclin-dependent kinase inhibitor p21cip1.     

The Helicobacter pylori UreI protein is not involved in urease activity but is essential for bacterial survival in vivo

We produced defined isogenic Helicobacter pylori ureI mutants to investigate the function of UreI, the product of one of the genes of the urease cluster. The insertion of a cat cassette had a strong polar effect on the expression of the downstream urease genes, resulting in very weak urease activity. Urease activity, measured in vitro, was normal in a strain in which ureI was almost completely deleted and replaced with a nonpolar cassette. In contrast to previous reports, we thus found that the product of ureI was not necessary for the synthesis of active urease. Experiments with the mouse-adapted H. pylori SS1 strain carrying the nonpolar ureI deletion showed that UreI is essential for H. pylori survival in vivo and/or colonization of the mouse stomach. The replacement of ureI with the nonpolar cassette strongly reduced H. pylori survival in acidic conditions (1-h incubation in phosphate-buffered saline solution at pH 2.2) in the presence of 10 mM urea. UreI is predicted to be an integral membrane protein and may therefore be involved in a transport process essential for H. pylori survival in vivo.     

Inhibitory effect of somatostatin on Helicobacter pylori proliferation in vitro

BACKGROUND & AIMS: Somatostatin regulates gastric function and cell proliferation. We investigated whether exogenous somatostatin modulates Helicobacter pylori proliferation in vitro. METHODS: Bacteria were cultured in 5 mL Brucella broth. Bacterial numbers of H. pylori (ATCC 43504) and Escherichia coli were calculated 48 and 5 hours after incubation, respectively, by counting the colonies on the blood agar. Chemicals were dissolved in absolute methanol and added to the broth at a final methanol concentration of 1%. Intrabacterial guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) levels were measured by radioimmunoassay. RESULTS: Somatostatin significantly suppressed H. pylori proliferation at levels at or above 10(-11) mol/L. A similar antiproliferative effect was observed with 8-bromo-cGMP. At concentrations at or above 10(-8) mol/L, dibutyryl cAMP slightly but significantly stimulated bacterial proliferation. Gastrin had no effect. Somatostatin antibody immunoglobulin G fraction blocked the antiproliferative effect of somatostatin on ATCC 43504. Scatchard plot showed that ATCC 43504 has one class of binding site with relatively high affinity (Kd, 0.31 nmol/L). Somatostatin at 10(-11) mol/L increased cGMP and cAMP in H. pylori 11-fold and 6-fold, respectively. In contrast, somatostatin neither bound E. coli nor affected its proliferation. CONCLUSIONS: Somatostatin, at a similar level in human gastric juice (approximately 10(-11) mol/L), suppresses H. pylori proliferation mediated in part by a cGMP-dependent pathway in vitro, indicating a possible inhibitory effect of somatostatin in the gastric lumen on H. pylori proliferation in humans.     

Reflux esophagitis; is the preventive eradication of Helicobacter pylori needed in patients on omeprazole?

The pattern of Helicobacter pylori gastritis depends on acid secretion. Profound acid suppressive therapy with proton pump inhibitors leads to a decrease of antral gastritis, but an increased severity of corpus gastritis. As such, maintenance therapy with these drugs for gastroesophageal reflux disease has consistently been associated with an increased incidence of atrophic gastritis in H. pylori infected patients. For this reason, the preventive effect of H. pylori eradication in these patients needs to be considered; this is being studied in prospective trials.     

Helicobacter mustelae and Helicobacter pylori bind to common lipid receptors in vitro

Helicobacter pylori is a recently recognized human pathogen causing chronic-active gastritis in association with duodenal ulcers and gastric cancer. Helicobacter mustelae is a closely related bacterium with similar biochemical and morphologic characteristics. H. mustelae infection of antral and fundic mucosa in adult ferrets causes chronic gastritis. An essential virulence property of both Helicobacter species is bacterial adhesion to mucosal surfaces. The aim of this study was to determine whether H. mustelae binds to the same lipids shown previously to be receptors for H. pylori adhesion in vitro. By using thin-layer chromatography overlay and a receptor-based enzyme-linked immunosorbent assay, H. mustelae was found to bind the same receptor lipids as H. pylori, namely, phosphatidylethanolamine and gangliotetraosylceramide. In addition, both H. pylori and H. mustelae bound to a deacylplasmalogen phosphatidylethanolamine. In contrast to H. pylori, H. mustelae binding to receptors was unaffected by motility or viability. Murine monoclonal and bovine polyclonal antibodies against exoenzyme S, and exoenzyme S itself (from Pseudomonas aeruginosa), inhibited binding of H. mustelae to phosphatidylethanolamine and gangliotetraosylceramide. These findings show that H. mustelae binds in vitro to the same lipid receptors as H. pylori and suggest that the adhesion of H. mustelae to such species is mediated by preformed, surface-exposed adhesins which include an exoenzyme S-like protein.     

Effect of genetic differences in omeprazole metabolism on cure rates for Helicobacter pylori infection and peptic ulcer

We report a 65-year-old woman with thyrotropin (TSH) secreting pituitary adenoma, who was diagnosed based on the lack of inhibition of serum TSH despite an increased serum free thyroxine (T4), a low response of serum TSH to thyrotropin releasing hormone, and a pituitary tumor as revealed by magnetic resonance imaging. The pituitary adenoma was, however, inoperable due to chronic respiratory failure. The treatment with octreotide in a dose of 100 microg b.i.d. resulted in inhibition of serum TSH and free T4 to euthyroid levels and considerable shrinkage of the pituitary tumor. These effects were continued over 8 months after the start of octreotide therapy without any adverse effects. These findings add further evidence that octreotide is useful for treating inoperable TSH secreting pituitary adenoma.     

US double-blind, controlled trials of omeprazole and amoxycillin for treatment of Helicobacter pylori

OBJECTIVE: To identify environmental and psychosocial factors associated with receiving special education services. DESIGN: The 1992 Minnesota Student Survey, an anonymous, self-report survey. SETTING: Minnesota public schools. PARTICIPANTS: A total of 121848 adolescents in the 6th, 9th, and 12th grades. MAIN OUTCOME MEASURES: Emotional status and potential environmental risk factors including family structure, family substance use problems, family violence, and sexual abuse were compared between adolescents reporting a history of having been in classes for learning problems and a grade- and race-matched comparison group of adolescents who had never been in classes for learning problems. Comparisons were conducted separately for male and female respondents. RESULTS: Compared with adolescents who had never been in classes for learning problems, a significantly greater proportion of male and female students who had been in special education classes lived in single-parent and nontraditional households, indicated that a family member had an alcohol or other drug problem, had witnessed or experienced physical abuse, and reported a history of sexual abuse and poor emotional health. Most of these associations remained significant when simultaneously controlling for the other factors in logistic regression. Significant factors showed modest odds ratios in the multivariate analyses (<1.6), except for the emotional status variable. Students with a history of receiving special education services had from 6 to 14 times the odds of reporting poor emotional health. This association was strongest among the youngest adolescents. CONCLUSION: Several environmental stressors and psychosocial factors, most notably poor emotional health, are associated with a history of special class placement for learning problems.     

Evaluation of a new urease reagent strip for detection of Helicobacter pylori in gastric biopsy specimens

OBJECTIVES: Determine sensitivity and specificity of a new urease reagent strip (URS) test for detection of Helicobacter pylori in gastric biopsy specimens. METHODS: Six paired biopsy specimens were obtained from the greater curvature of the distal antrum, the lesser curvature near the incisura, and the corpus along the greater curvature during 66 procedures on 59 patients with endoscopic findings of gastric antral mucosal erythema or erosions, or gastric or duodenal ulcers. One biopsy from each site was tested with the URS. The second was evaluated with histology. A final antral biopsy was evaluated with a urea/gel test. RESULTS: Twenty-eight of the 66 cases were histologically positive, with H. pylori observed in at least one of the three biopsy sites. The URS test correctly identified all 28. Of 193 individual biopsy specimens, 78 were positive for H. pylori. The URS correctly identified 77. Sensitivity was 0.99, specificity 0.95, positive predictive value 0.93, negative predictive value 0.99, and kappa 0.92. Average time to positive was 20 min. Twenty-seven antral biopsies were histologically positive for H. pylori. The URS test correctly identified all 27, whereas the urea/gel test correctly identified 21. For antral sites, URS sensitivity was 1.00 and specificity 0.95 versus urea/gel test sensitivity of 0.75 and specificity of 1.00. CONCLUSIONS: In this sample, the URS test is as accurate as histology in diagnosing H. pylori infections, and it provides results in less time and at a lower cost than histology.     

Helicobacter pylori does not migrate from the antrum to the corpus in response to omeprazole

BACKGROUND: Omeprazole is known to have an effect on Helicobacter pylori in vivo. One opinion is that H. pylori "migrates" from the antrum to the corpus in response to omeprazole therapy. METHODS: To determine whether H. pylori migrates in response to omeprazole, we assessed the presence of H. pylori in the antrum and corpus in duodenal ulcer patients receiving omeprazole for 4 wk. Culture and histological examination of antral biopsies (Genta stain) were performed before patients received omeprazole, at the end of therapy, and 4-6 wk later. The end points were presence or absence of H. pylori and the number of H. pylori colonies per biopsy. RESULTS: Seventy-two patients had H. pylori in both the antrum and corpus at entry and 4-6 wk after ending therapy. Three general patterns were prevalent at the end of omeprazole therapy: antrum- and corpus-positive (54%), antrum-negative and corpus-positive (24%), both antrum- and corpus-negative (21%), and one patient had antrum-positive with corpus-negative (1%). Evaluation of the number of colonies per biopsy in those who remained H. pylori-positive in both the antrum and corpus throughout showed that the number of H. pylori decreased in both the antrum and corpus during therapy (507 +/- 60 vs. 225 +/- 51, p < 0.01 and 415 +/- 58 vs. 290 +/- 46 0.1) for antrum and corpus, respectively, and tended to return to pre-therapy levels 4-6 wk later. The number of H. pylori in the corpus also decreased in the antrum-negative and corpus-positive group during therapy with omeprazole (433 +/- 87 vs. 185 +/- 61, p < 0.05). In most of the patients studied, the number of H. pylori in the corpus was less posttreatment than it was pretreatment. The decrease in H. pylori load was also reflected in the development of false-negative urea breath tests. CONCLUSIONS: Omeprazole is detrimental to H. pylori in both the antrum and the corpus; migration from the antrum to the corpus in response to omeprazole is a myth.     

Evaluation of a new reagent strip rapid urease test for detection of Helicobacter pylori infection

BACKGROUND: Rapid urease tests are commonly used as a convenient method to detect Helicobacter pylori infection. Our previous experiments demonstrated enhanced efficacy of agar gel rapid urease test compared with reagent strip rapid urease tests. We evaluated the efficacy of PyloriTek, a new reagent strip rapid test for detecting H. pylori infection. METHODS: Gastric antral mucosal biopsy specimens were obtained for comparison between agar gel rapid urease tests and PyloriTek (200 specimens). The rapid urease test to be used first was selected randomly. H. pylori status was determined using the Genta stain. Culture was performed to confirm H. pylori status when false rapid urease tests were suspected. RESULTS: One hundred patients were studied; 68 had H. pylori infection. There were two false-negative and one false-positive PyloriTek when scored at 1 hour, compared with only one false-positive and no false-negative tests at 2 hours. With the agar gel rapid urease tests, there were no false-positive tests and 5 false-negative tests when scored at 1 hour, 2 false-negative tests at 12 hours and 1 at 24 hours; there were no false-positive tests. At 1 hour, 3% (95% CI = 1% to 9%) of PyloriTek tests had an erroneous categorization of H. pylori status compared with 5% for the agar gel rapid urease tests (95% CI = 1.6% to 11%) (p > 0.7). CONCLUSION: The new reagent strip rapid urease test, PyloriTek, is rapid and comparable in accuracy to agar gel rapid urease tests for detecting H. pylori Infection.