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Helicobacter pylori, a gram-negative neutralophile, is the only organism known to colonise the normal acid-secreting human stomach. About 50% of the world's population is infected. Colonisation is associated with several gastric diseases, including gastritis, peptic and duodenal ulcers, gastric carcinoma and MALT lymphoma.[2-5] In 1994, the WHO classified the organism as a type 1 carcinogen. Despite recent findings that H. pylori infection may have a beneficial effect on gastroesophageal reflux disease (GERD) and other nongastric manifestations of infection, the consequences of gastric colonisation alone argue for its eradication.[7, 8]
Standard H. pylori eradication therapy involves a complicated regimen of at least two antibiotics (clarithromycin and amoxicillin or metronidazole) and a proton pump inhibitor or antibiotics, acid suppression and bismuth.[9, 10] It has never been clear as to why two antibiotics, one targeting cell wall biosynthesis and the other targeting protein synthesis, are required for eradication. Acid suppression clearly improves the efficacy of antibiotic treatment.
Emerging antibiotic resistance to clarithromycin has made successful treatment of infection progressively more difficult, with the success rate of standard triple therapy now at 70%, well below the 80% required for treatment of infectious diseases. Alternative eradication regimens have been suggested, such as sequential therapy (PPI and amoxicillin for 5 days followed by PPI, clarithromycin and metronidazole) or concomitant therapy, but the results of these regimens are not comparable to the initial eradication results with triple therapy (~95%). Quadruple therapy (addition of colloidal bismuth subcitrate to standard triple therapy regimens) has shown promise, with cure rates currently between 80% and 90%.[9, 12] However, as bismuth containing quadruple therapies also rely on antibiotics, this regimen is sensitive to and contributes to increasing antibiotic resistance. Therefore, it is likely that eradication rates will decrease with this regimen as it has for triple therapy.
There have been several hypotheses as to the role of PPIs in eradication therapy. A direct effect of the PPI on the viability of the organism in vitro has been suggested. However, this required the administration of very high doses of the PPI to show direct inhibition. At these concentrations, there is acid-independent thiol reactivity of the PPI, resulting in nonspecific effects. Another hypothesis is increased gastric bioavailability of amoxicillin after PPI administration, but it is generally agreed that gastric acidity does not influence the pharmacokinetics of this antibiotic.
Helicobacter pylori resistance to amoxicillin is rare, suggesting it would be the antibiotic of choice as long as the bacteria remain susceptible to its action. Amoxicillin acts by inhibiting the synthesis of the bacterial cell wall. It inhibits cross-linking between the linear peptidoglycan polymer chains that make up a major component of the cell wall. Therefore, bacteria must be actively dividing and synthesising the cell envelope for this antibiotic to be effective. In the absence of urea, H. pylori enters a nonreplicative, but viable state when the environmental pH is less than 6.0 and greater than 3.0.[16, 17] In vivo gene expression of H. pylori indicates that the average pH at the site of infection in the gerbil stomach is about 3.5, placing the organism in a nondividing state, decreasing the efficacy of amoxicillin for eradication. Thus, increasing the intragastric pH to >5.0 should induce the bacteria to enter the replicative state and become more susceptible to both amoxicillin and to the protein synthesis inhibitor, clarithromycin.
Herein, we explore the effects of lengthy exposure of the organisms to pH 7.4, 4.5 and 3.0 in the presence of 5 mM urea, taking advantage of a method developed in our laboratory that exposes a small volume of the bacteria in a dialysis chamber to a large volume of pH-adjusted medium without added buffer, allowing analysis for 4, 8 or 16 h. Analysis of the bacterial transcriptome at pH 3.0 reveals down regulation of ~400 genes, including genes encoding envelope synthesis, cell division and protein synthesis. Analysis of viability and growth potential shows that cell viability is decreased at pH 3.0, but not at higher pH, and this pH also decreases the number of colony forming units (CFUs). Also, at pH 3.0, in contrast to the higher pH levels, the bactericidal effect of ampicillin disappears, indicating that the lack of growth at pH 3.0 attenuates penicillin sensitivity.
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This study shows that at pH 3.0, H. pylori is viable, but in a nonreplicative state, diminishing the bactericidal efficacy of penicillin antibiotics and also that of protein synthesis inhibition by clarithromycin. This is supported by the large number of cell envelope and cell division genes down regulated at pH 3.0 and the loss of ampicillin efficacy. Induction of profound acid inhibition using potent H, K-ATPase antagonists, which raise the intragastric pH to between 5.0 and 7.0, should stimulate growth of H. pylori and increase the bactericidal effect of amoxicillin, which should result in eradication.
There has been considerable controversy as to the pH of the gastric habitat of H. pylori. It has been proposed that there is a gastric barrier to proton back diffusion from the gastric lumen to the gastric surface due to both mucus and bicarbonate secretion. However, recent experiments using fluorescent dyes or microelectrodes in infected mice show that the pH gradient collapses when the luminal pH falls to <3.0.[24, 25] Also, analysis of the transcriptome of bacteria isolated from the gerbil stomach in comparison with the pH dependent transcriptome in vitro showed that average pH in their gastric habitat was about pH 3.5.
The level and duration of acid suppression affects the success of eradication with PPIs.[26-28] Nocturnal acid breakthrough is a factor in the failure of triple therapy. The major location of bacteria resistant to treatment is the more acidic fundus, and hence the lower pH in this region promotes H. pylori growth inhibition and contributes to the failure of triple therapy.
Improved inhibition of acid secretion increases the rate of eradication. Cure rates of standard triple therapy depend on the efficacy of PPI-dependent inhibition of acid secretion. Although PPIs are covalent inhibitors of the gastric H, K-ATPase, their short plasma half-life prevents adequate elevation of pH during the night, when newly synthesised pumps are not exposed to the PPI. This, coupled with the slow growth rate of H. pylori, results in insensitivity of the organisms to growth dependent antibiotics, as the pH falls frequently to <3.0. In a recent study, q.d.s. omeprazole, dosed to maintain intragastric pH above pH 5.5 for 16 h, and amoxicillin dual therapy eradicated H. pylori infection, substantiating the idea that improved acid inhibition would improve results of eradication therapy.
Further evidence that increased PPI dwell time, and therefore better acid inhibition, improves H. pylori eradication comes from studies on slow omeprazole metabolisers. PPIs are mainly metabolised by cytochrome 2C19 (CYP2C19), and in patients who are homozygous for the loss of CYP2C19, leading to slower metabolism and increased plasma dwell time of the PPI, inhibition of acid secretion is significantly increased. These patients were successfully treated for H. pylori eradication with omeprazole and amoxicillin alone.[31, 32] Recently, it was shown that 10 mg rabeprazole q.d.s. maintained plasma PPI levels above the threshold level required for H+,K+-ATPase inhibition for 24 h, resulting in a median intragastric pH of 6.6 independent of CYP2C19 status. These results show that increased acid inhibition by PPIs may enable dual therapy, and this can be achieved by increasing the amount of time that this type of drug is in the blood. However, there have been several attempts at dual therapy with lack of success, likely due to inadequate elevation of pH during the night.[34, 35]
There are several PPIs, which would likely raise intragastric pH close to neutrality, tenatoprazole, a slowly metabolised PPI, AGN904, an omeprazole prodrug with a longer dwell time than omeprazole, and the potassium-competitive acid blockers TAK-438 and AZD0865.[36-40]
The data presented herein demonstrate that an improvement in acid inhibition extending into night time hours, maintaining the intragastric pH close to neutrality without acidic excursions overnight, would greatly improve eradication rates of triple therapy and perhaps allow dual therapy with a potent H, K-ATPase inhibitor and amoxicillin. These results provide a template for new clinical studies on H. pylori eradication.