Effect of proton pump inhibitor on the movement of Helicobacter pylori across the mucus layer to the gastric lumen

Background: Helicobacter pylori ( H. pylori ) cannot usually be detected in the gastric juice and it is thought that H. pylori may reside under the mucus layer for long term. The mechanisms of action of proton pump inhibitor (PPI)for H. pylori eradication are not entirely clear. Our study aimed to determine the role of PPI on the movement of H. pylori across the mucus layer to the gastric lumen and the mechanism of PPI on H. pylori eradication. Methods: Patients with H. pylori infection were intravenous injected with PPI (intervention group, n=31) or without PPI (control group, n=37). The presence of H. pylori in the gastric juice was evaluated by the rapid urease test (RUT), polymerase chain reaction (PCR), and culture methods. Results: The H. pylori positive detection rates were all significantly higher among patients in the intervention group than among patients in the control group by the RUT ( P < 0.0001), PCR ( P < 0.0001), and culturing ( P = 0.0386). Conclusion: H. pylori can penetrate across the mucus layer to the gastric lumen following PPI intervention, and thus it might represent a novel target in the eradication of H. pylori .

gastric pathogens 4-6 causing chronic gastritis, functional dyspepsia, peptic ulcer, gastric adenocarcinoma, and lymphoma. 7,8 It has also been found to be associated with multiple extra gastrointestinal diseases, such as cardiovascular diseases, hematological system diseases, diabetes, and immune diseases. In 1994, the International Agency for Research on Cancer consensus group listed H. pylori as a class I human carcinogen. 9 The eradication of H. pylori is a major global public health issue. Currently, several diagnostic tests are available for determining the presence of H. pylori, such as rapid urease test (RUT), histology, polymerase chain reaction (PCR), culture, urea breath test (UBT), and serology.
At present, the standard method for the eradication of H. pylori is triple (proton pump inhibitor [PPI] + two kinds of antibiotics) or quadruple (PPI + two kinds of antibiotics + bismuth) therapy based on PPI recommended by the international guidelines from the Maastricht V consensus, 10 the Toronto consensus, 11 and other consensuses from many countries. [12][13][14][15] However, using this traditional treatment, it has become increasingly difficult to eradicate H. pylori because of the side effects and increasing antibiotic resistance. 16,17 The search for alternative treatments, such as microecological agents, traditional Chinese medicine, second-line antibiotics (such as Rifabutin), and vaccines 18 has become a particular focus.
However, H. pylori eradication remains a serious challenge. Until now, the mechanism of action of PPI, antibiotics, and bismuth for H. pylori eradication has not been fully understood. A new understanding of the H. pylori eradication mechanism is needed to tailor accurate and effective H. pylori eradication therapy.
H. pylori can invade into the surface of the gastric mucosa and penetrate across the mucin layer. We therefore hypothesized that H. pylori may also penetrate across the 5 mucin layer to the gastric lumen. In this study, we investigated the movement of H. pylori up and down the gastric mucus layer and analyzed the effect of PPI on the movement of H. pylori. .

Ethical considerations
The Esophagogastroduodenoscopy was performed after an 8 h fast without any 6 defrother, anesthetic or other orally-administered drugs. Patients lay on the left recumbent position of the examining table and were successfully anesthetized by intravenous injection of propofol. Then 2-3 ml of fasting gastric juice not containing blood, bile or any other components were sampled from the fundus/corpus at endoscopy by means of a sterile cannula with an external connection to a 10 ml sterile syringe. The pH of the gastric juice was determined and a Giemsa stain was performed. The gastric fluid was dropped into a RUT reagent bottle (SanQiang Biological and Chemical Co. Ltd., Fujian, China) and was observed for at least 30 min at room temperature. After each examination, the endoscope was washed with 2% glutaraldehyde and disinfected with 70% ethanol followed by rinsing with sterile water. The sterile cannula was reformed from a sprinkler tube by cutting off its front end, which was then washed with sterile water, treated with an enzymatic hydrolysate, and doused in glutaraldehyde for at least 10 h, followed by rinsing with sterile water after each examination.

Bacterial cultivation
Culturing of the gastric fluid samples to detect H. pylori was performed using the centrifuge tube, add 50u1 purified water and 50μl DNA extract reagent into the centrifuge tube and mix it thoroughly, and then centrifuge it at 12,000 RPM for 5 minutes. Specific primers and fluorescent probes for the highly conserved coding region of H. pylori genome as the target region were included in this kit. The reaction was performed using the ABI 7500 detection system (Applied Biosystems, Foster, CA, USA) with preliminary denaturation for 8 min at 50°C, 2 min at 93°C, followed by 10 amplification cycles of denaturation at 93°C for 45 s and annealing at 55°C for 1 min, followed by 30 amplification cycles of denaturation at 93°C for 30 s, annealing at 55°C for 45 s, and primer extension at 55°C for 45 s, with a fluorescence acquisition step at the end of the extension. The reference value for this kit was 27.02, which was determined by the ROC curve method. Samples were considered to be H. pylori positive when the amplification curve was of typical 'S' type and the Ct value was ≤ 27.02.

Statistical analysis
Calculations were performed using SPSS software version 22(IBM Corp., Armonk, NY, USA). Descriptive analysis was performed for demographic and clinical features.
Results are presented as the mean ± SD for quantitative variables and the number (percentage) for qualitative variables. The Chi-square test was used to compare categorical data. The T-test was used to compare normally distributed continuous variables. P < 0.05 indicated a statistically significant difference. Table 1 presents the patients' characteristics. The gastric fluid was collected from 68 patients infected with H. pylori after the gastric fluid mixed with blood or bile excluded. The pH value of the gastric juice was significantly higher in the intervention group than in the control group (P < 0.0001).   The positive frequency was higher in the intervention group than in the control group (P < 0.0001) (Fig. 4).

Discussion
H. pylori is a Gram-negative microaerobic bacterium that is spiral in shape and has 9 2-6 polar flagella for mobility. This bacterium resides between the surface of the gastric mucosa and the mucin layer without evidence of intracellular parasitism. 19 H. pylori can only survive at a periplasmic pH of 4.0-8.5 and can only grow at a periplasmic pH of 6.0-8.5. It is difficult to detect H. pylori in the gastric lumen because it is such an acidic environment.
The human stomach is divided into three anatomic regions: the cardia, the fundus/corpus, and the antrum. The antrum secretes alkaline mucus 4-5 cm around the antrum. Therefore, H. pylori is mainly distributed in the antrum. 20 H. pylori distribute in two ways: (1) by colonizing the surface of the gastric pit and epithelial cells, and (2) by colonizing above the tissue surface mucus layer. The latter is more common. An animal model revealed that H. pylori colonizes a zone 0-25 μm above the tissue surface mucus layer, to a total thickness of about 100 μm. 21 While the gastric lumen has a pH of 1-2, a pH gradient exists across the mucus layer, reaching a pH of 6-7 at the surface of the mucosa. 22 Using chemotaxis, H. pylori navigate this pH gradient to reach their niche environment near the host epithelium. 23,24 Research has revealed that the stomach supports a bacterial community comprising hundreds of phylotypes, 25, 27 while a pH of < 4 prevents bacterial overgrowth. It was reported that the microbial density in the stomach is 10 1 -10 3 CFU/g 28,29 This high density of bacteria means that Giemsa staining is not an optimal method for studying H. pylori in the stomach. Therefore, in this study, we employed the RUT, PCR analysis, and culture methods to study H. pylori in gastric juice.
Until now, little is known about the movement characteristics of H. pylori all over the world. We use PPI intervene the patients infected with H. pylori and study the movement characteristics of H. pylori. In this study, the pH of gastric juice in control group patients was range from 1 to 3 with an average of 1.59, which was consistent with normal pH value of gastric juice. The pH of the gastric juice of patients in the intervention group was higher than that in the control group, but H. pylori was detected in both groups. Culture is the gold standard method for detecting the presence of viable H. pylori. In our study, the positive detection rate was significantly higher in the gastric juice of patients in the intervention group than of patients in the control group. The RUT and PCR analysis also indicated that positive detection rates were significantly higher among patients in the intervention group than among those in the control group. The results of culturing, the RUT, and PCR were consistent. Our findings indicated that PPI intervention may induce H. pylori to penetrate across the mucus layer from the surface of the gastric mucosa to the gastric lumen. However, one positive case was detected by the RUT of the gastric fluid from the control group and this sample had a pH value of 1, possibly indicating that the gastric fluid may be mixed with slight bile. In addition, six samples from the control group tested positive by PCR, which might indicate that there was a high load of H. pylori for these bacteria to be detected in the gastric lumen.
H. pylori is a fastidious microorganism that requires complex growth media. The H. pylori living environment is small in the mucus layer and a key feature of this bacterium is its microaerophilicity, with optimal growth at O 2 levels of 2% to 5%. 30 H. pylori is unable to survival under normal atmospheric conditions or under absolute anaerobic conditions. H. pylori requires complex growth media rich in nutrients. However, the mucus layer has low permeability to most molecules such as protons, O 2 , and nutrient macromolecules. If the condition of juice in gastric lumen met the qualification mentioned above, H. pylori may penetrate across the mucus layer to the gastric lumen.
It is reported that PPI alone also exerts direct antimicrobial activity against H. pylori with 6%-7.7% eradication rate, 31,32 which was confirmed by Meining and colleagues. 33 Iwahi and coworkers 34 35 Motile bacteria sense chemical gradients by means of chemoreceptor proteins, such as BabA, with a pH-sensor mechanism 36 , and relay the information to the flagellar motor 37 to direct movement toward an environment with optimal concentrations of both electron acceptors and proton/electron donors.
Another study reported that among H. pylori exposed to neutral pH, only about 7% of the culture were motile and traveled at an average speed of 10.5 μm per s. By contrast, among cells that were shifted to an acidic pH, 66% were motile with a significantly faster average speed of 24.3 μm per s 38 . It seems likely that H. pylori uses the pH-sensing mechanism for orientation along the transmucus pH gradient.

Availability of data and material
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The    The movement characteristics of H. pylori under PPI intervention and the mechanisms of acti