The highly motile human pathogen Helicobacter pylori lives deep in the gastric mucus layer. To identify which chemical gradient guides the bacteria within the mucus layer, combinations of luminal perfusion, dialysis, and ventilation were used to modify or invert transmucus gradients in anaesthetized Helicobacterinfected mice and Mongolian gerbils. Neither changes in lumen or arterial pH nor inversion of bicarbonate͞CO2 or urea͞ammonium gradients disturbed Helicobacter orientation. However, elimination of the mucus pH gradient by simultaneous reduction of arterial pH and bicarbonate concentration perturbed orientation, causing the bacteria to spread over the entire mucus layer. H. pylori thus uses the gastric mucus pH gradient for chemotactic orientation. Helicobacter pylori, a motile Gram-negative human pathogen that causes gastritis and duodenal and gastric ulcers and increases the risk of gastric cancer (1), lives within the gastric mucus layer. The majority of bacteria are located deep in the mucus, close to the surface of the epithelium. The mucus is continuously secreted in the glands and by surface epithelial cells and is degraded at the luminal surface (2). Because of this rapid mucus turnover, the bacteria need motility and spatial orientation to avoid being carried into the lumen, where the acidic pH inhibits growth and paralyzes motility (3, 4). Orientation therefore plays a central role in acute colonization and the chronic persistence of H. pylori.Motile bacteria sense chemical gradients by means of chemoreceptor proteins and relay the information to the flagellar motor (5). In the case of H. pylori, it must be a chemical gradient in the gastric mucus layer that guides the bacteria. Within the mucus layer, diffusion is effectively restricted, so that the concentration of most substances entering the mucus from the epithelial surface will remain constant through the entire width of the layer. Despite this, at least three chemical gradients are known to exist here: a proton (pH) gradient (2, 6), a bicarbonate͞CO 2 gradient (7,8), and, in the Helicobacter-infected mucosa, a urea͞ammonium gradient (9) created by bacterial urease. We hypothesized that H. pylori may use one or several of these transmucus gradients for orientation in vivo.We have previously developed a method to precisely determine the distribution and colonization density of Helicobacter felis in the gastric mucus of mice in vivo (3). This method uses a micromanipulator-controlled sampling device (10) to extract nanoliter samples from the mucus and mucosa of anesthetized animals. After immediate immobilization of the bacteria by cooling, the density and distribution of the bacteria can be studied by digital image processing.To identify gradients involved in the orientation of Helicobacter spp., we inverted or modified individual gradients in anesthetized H. pylori-infected Mongolian gerbils or H. felisinfected mice and studied the effect of these changes on the distribution of bacteria in the mucus layer. The data show that gastric Helicob...

The human pathogen Helicobacter pylori has infected more than half of the world's population. Nevertheless, the first step of infection, the acute colonization of the gastric mucus, is poorly understood. For successful colonization, H. pylori must retain active motility in the gastric lumen until it reaches the safety of the mucus layer. To identify the factors determining the acute colonization, we inserted bacteria into the stomach of anesthetized Mongolian gerbils. We adjusted the gastric juice to defined pH values of between 2.0 and 6.0 by using an autotitrator. Despite the fact that Helicobacter spp. are known to survive low pH values for a certain time in vitro, the length of time that H. pylori persisted under the assay conditions within the gastric juice in vivo was remarkably shorter. In the anesthetized animal we found H. pylori to be irreversibly immotile in less than 1 min at lumen pH values of 2 and 3. At pH 4 motility was lost after 2 min. However, the period of motility increased to more than 15 min at pH 6. Blocking pepsins in the gastric lumen in vivo by using pepstatin significantly increased the period of motility. It was possible to simulate the rapid in vivo immotilization in vitro by adding pepsins. We conclude that pepsin limits the persistence of H. pylori in the gastric chymus to only a few minutes by rapidly inhibiting active motility. It is therefore likely that this short period of resistance in the gastric lumen is one of the most critical phases of Helicobacter infection.

Gastroesophageal reflux induced by relaxation of the lower esophageal sphincter was shown to reach the middle ear in our Mongolian gerbil model. These results support recent hypotheses linking gastroesophageal reflux to the development of otitis media with effusion.

The risk of Helicobacter pylori infection is highest in childhood, but the colonization process of the stomach mucosa is poorly understood. We used anesthetized Mongolian gerbils to study the initial stages of H. pylori colonization. Prandial and postprandial gastric conditions characteristic of humans of different ages were simulated. The fraction of bacteria that reached the deep mucus layer varied strongly with the modelled postprandial conditions. Colonization success was weak with fast gastric reacidification typical of adults. The efficiency of deep mucus entry was also low with a slow pH decrease as seen in pH profiles simulating the situation in babies. Initial colonization was most efficient under conditions simulating the postprandial reacidification and pepsin activation profiles in young children. In conclusion, initial H. pylori colonization depends on age-related gastric physiology, providing evidence from an in vivo infection model that suggests an explanation why the bacterium is predominantly acquired in early childhood.

The gastric lumen represents a bactericidal barrier, whose major components are an acidic pH and a family of isoenzymes of the gastric aspartate protease, pepsin. To evaluate whether specific pepsins are specialized in antibacterial protection, we tested their effects on the gastric pathogen Helicobacter pylori. In a recent study we found pepsin to affect the motility of the bacteria, one of its most important virulence factors. We were able to show that the antibacterial effect of pepsin occurs in two phases: rapid loss of motility and subsequent destruction. In the present study we used the rapid pepsin-induced bacterial immobilization as a marker of antibacterial efficiency. The proteolytic activity of different pepsins was normalized to values between 2 and 200 U/ml in the hemoglobin degradation test of Anson, performed at pH 2 and 5. We found that pepsin C completely inactivates H. pylori at proteolytic activities of 2 (pH 5) and 20 (pH 2) U/ml. In contrast, the activities of pepsin A and chymosin required to affect Helicobacter motility were ten times higher.

Until now, it has been unclear how proton pump inhibitors (PPIs) support Helicobacter pylori therapy. We tested whether the PPI omeprazole acts on the spatial orientation of H. pylori in the gastric mucus of infected Mongolian gerbils. Following repetitive PPI administration once daily but not following single doses or administration every 8 h, the bacterial spatial distribution changed, indicating a loss of orientation. Therefore, the therapeutic scheme of PPI administration may affect efficiency of treatment.The gastric pathogen Helicobacter pylori infects about half of all humans (15, 22) and causes ulcers (14) and gastric adenocarcinomas (4, 7). To cure H. pylori infection, a combined treatment with antibiotics and a proton pump inhibitor (PPI) for gastric acid suppression is used. Despite frequent administration in conventional triple therapies (12) and the sequential therapies currently being developed (8, 9, 13, 23), the effect of PPIs in therapy for this infection is poorly understood. A finding of major importance was that combined treatment with a PPI causes an increased concentration of the regularly used antibiotic clarithromycin in the gastric mucus (11). Due to the interdependent regulation of acid and mucus secretion, a PPI may reduce mucus secretion (10). In the case of substances with low gastric clearance rates, decreased mucus secretion after administration of a PPI could enhance their concentrations in the mucus. However, this observation does not explain the mechanism of action of PPIs, since antibiotic schemes without the macrolide clarithromycin (using, e.g., the deeply penetrating fluoroquinolone moxifloxacin [1,16]