A new understanding of the H. pylori eradication mechanism

Background: Helicobacter pylori (H. pylori) cannot usually be detected in the gastric juice and it is thought that H. pylori may be implanted under the mucus layer for long term. The mechanisms of action of proton pump inhibitor (PPI), antibiotics, and bismuth 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, antibiotics, and bismuth 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. The direct antimicrobial activity of PPI might because of diminished numbers of H. pylori due to probiotics in the gastric lumen. Antibiotics and bismuth might play a local sterilization role in the gastric lumen when H. pylori penetrate across the mucus layer.


Introduction
Helicobacter pylori ( H. pylori) is one of the most common bacterial infections, potentially lasting for decades in an individual, and infection can lead to multiple diseases. The prevalence of H. pylori infection is higher than 50% in much of the world, and ranges from 41.35% to 72.3% in China, with an average of 56.22%. 1 H. pylori is widely regarded as one of the most common gastric pathogens 2-4 causing chronic gastritis, functional dyspepsia, peptic ulcer, gastric adenocarcinoma, and lymphoma. 5,6 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. 7 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 the rapid urease test (RUT), histology, polymerase chain reaction (PCR), culture, the 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, 8 the Toronto consensus, 9 and other consensuses from many countries. [10][11][12][13] However, using this traditional treatment, it has become increasingly difficult to eradicate H. pylori because of the side effects and increasing antibiotic resistance. 14,15 The search for alternative treatments, such as microecological agents, traditional Chinese medicine, second-line antibiotics (such as Rifabutin), and vaccines 16 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 implant into the surface of the gastric mucosa and penetrate across the mucin layer. We therefore hypothesized that H. pylori may also penetrate across the 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 in detail the mechanisms of current H. pylori eradication therapy.

Ethical considerations
The Esophagogastroduodenoscopy was performed after an 8 h fast without any 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 ofthegastric fluid samples to detect H. pylori was performed using the

Results
Patients' characteristics 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).

Discussion
H. pylori is a Gram-negative microaerobic bacterium that is spiral in shape and has 2-6 polar flagella for mobility. This bacterium implants between the surface of the gastric mucosa and the mucin layer without evidence of intracellular parasitism. 17  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. 18 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. 19 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. 20 Using chemotaxis, H. pylori navigate this pH gradient to reach their niche environment near the host epithelium. 21,22 Research has revealed that the stomach supports a bacterial community comprising hundreds of phylotypes, 23-25 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. 26,27 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 too high a 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 bacteriumis its microaerophilicity, with optimal growth at O 2 levels of 2% to 5%. 28 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.
In recent years, the application of probiotics in the eradication of H. pylori has become an area of increasing research interest. Probiotics are capable of influencing bacterial growth by secreting antibacterial substances, and their metabolites may diminish the number of H. pylori. 29 Lactic acid might have an additional effect on H. pylori by inhibiting urease and lowering the pH 30 (Fig. 5). The Maastricht V consensus 8 reported that several probiotics had been administered combined with antibiotic therapies to treat H. pylori infection and probiotics could significantly increase the treatment efficacy of triple therapy. 31 It is reported that PPI alone also exerts direct antimicrobial activity against H. pylori with a 6%-7.7% eradication rate, 32,33 which was confirmed by Meining and colleagues. 34 Iwahi and coworkers 35 practiced a more in-depth study of PPI, and surprisingly found that lansoprazole could inhibit the growth of H. pylori in vitro, similar to the antibacterial effects of antibiotics. A series of subsequent studies found that omeprazole, pantoprazole, and rabeprazole have different degrees of inhibitory effects on H. pylori in vitro, but the degree of bacteriostasis of PPIs in vitro differs greatly, with rabeprazole showing the strongest bacteriostatic effect. However, these differing effects of PPIs have not been reported in clinic. According to our research results, we propose that PPI intervention may reduce the pH gradient, disturbing the pH chemotaxis of H. pylori and leading to the penetration of the mucus layer. Under this mode of movement, H. pylori might be diminishedbythe presence ofprobiotics above the mucus layer. Otherwise, when the pH value in the gastric lumen decreases below 4, H. pylori would return to thehost epithelium, again being exposed to the deleterious effects of probiotics. In addition, the high pH of the gastric juice in the lumen can accelerate the growth of probiotics to assist with the eradication of H. pylori.
The flagella of H. pylori often carry a distinctive bulb at the end. The flagella confer motility and allow rapid movement in viscous solutions such as the mucus layer overlying the gastric epithelial cells. 36 Motile bacteria sense chemical gradients by means of chemoreceptor proteins, such as BabA, with a pH-sensor mechanism, 37 and relay the information to the flagellar motor 38 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. 39 It seems likely that H. pylori uses the pH-sensing mechanism for orientation along the transmucus pH gradient. H. pylori can penetrate across the mucus layer to the gastric lumen when the pH gradient is reduced by PPI intervention.
Until now, the mechanism by which antibiotics eradicate H. pylori has been controversial.
It was thought that the drug was delivered directly following oral administration, or indirectly following intestinal absorption, and transferred from the blood into the stomach across the gastric mucosa. Traditionally it has been thought that drugs need to penetrate across the mucus layer from the gastric lumen to the epithelial surface, or vice versa, to reach the target bacteria, but delivery is limited by the permeability of the mucus layer. In its physiological state, the gastric mucosa has low permeability to most molecules, from protons to macromolecules. 40,41 Unlike previous reports, our findings indicated that antibiotics play a local function in the gastric lumen when H. pylori penetrates across the mucus layer to the gastric lumen because of the use of PPI (Fig. 5). The "battlefield" that we originally considered that antibiotics eradicated H. pylori changed from the submucous layer to the gastric lumen. Therefore, current concept of H. pylori should been changed.
Bismuth-based quadruple therapy has been used as first-line therapy and has shown excellent effects in H. pylori eradication even for antibiotic resistance strains. [42][43][44][45][46] However, the mechanism of action of bismuth drugs is not fully understood. 46 Bismuth agents can form a bismuth complex with glycoproteins, which form a diffuse barrier to acids. It has been shown that bismuth also has bactericidal activity. When H. pylori penetrates across the mucus layer to the gastric lumen under PPI intervention or returns to thehost epithelium, bismuth may damage, or even eradicate, H. pylori.

Conclusions
Our study indicated that H. pylori can penetrate the mucus layer and enter the gastric lumen where it acquires O 2 and nutrients under PPI intervention. The mechanism of direct antimicrobial activity of PPI may be that PPI disturbs the pH chemotaxis of H. pylori, leading to penetration across the mucus layer, where it is diminishedbyprobiotics above the mucus layer and eliminated by gastric emptying. Antibiotics and bismuth may play a local sterilization role in the gastric lumen when H. pylori penetrates across the mucus layer. Through the movement characteristic of H. pylori, we provide reliable data for the optimal application time of probiotics, antibiotics and bismuth as well as a theoretical basis for improving the eradication of H. pylori.        Comparison of gastric fluid PCR for H. pylori with and without PPI. The positive frequency was higher in PPI intervention group than that in Control group (P < 0.001).

Figure 5
The movement characteristics of H. pylori under PPI intervention and the mechanisms of action of PPI, antibiotics and probiotics.