H. pylori eradication
Mechanism of the suppressive effect of probiotics on H.pylori: The major mechanisms of probiotics against H. pylori infection are thought to be competition with H. pylori for binding sites on gastric epithelial cells, reinforcement of the mucosal barrier, and secretion of bactericidal organic acids (e.g., lactic acid). These are the principle anti-bacterial effects exerted by probiotics. As for the mechanism of competitive binding, L. reuteri is reported to inhibit the attachment of H. pylori on the epithelial cell surface by competitive binding to asialo-GM1 and surface receptors. Moreover, other probiotic species (e.g., L. acidophilus, L. johnsonii and L. salivarius) were reported to prevent H. pylori colonization through specific adhesion molecules[25,26]. Specific binding of H. pylori to the host cell then induces the production of IL-8 through the type IV secretion system. Tamura et al also demonstrated competition for binding sites between H. pylori and a probiotic strain LG21 using a coculture system with MKN45 cells (a human gastric epithelial cell line) and H. pylori. Large amounts of IL-8 were produced in the gastric epithelial cells cocultured with H. pylori (106 CFU) alone. When 106 CFU of non-treated live LG21 (equivalent to the number of H. pylori) was added to the coculture system, the amount of IL-8 secreted into the culture supernatant significantly decreased. However, UV- or heat-treated LG21 could not exert any suppressive effect on H. pylori-induced IL-8 production, even at 108 CFU (100 times the amount of non-treated LG21). An adherence assay in their study supported that LG21 competitively inhibited the binding of H. pylori to MKN45 cells, which suppressed the production of IL-8. Moreover, they demonstrated that the suppressive effect of LG21 also worked in the human stomach. The measurement of the IL-8 Level in gastric biopsy specimens from H. pylori-infected subjects also revealed that the oral intake of probiotic LG21 significantly suppressed the generation of IL-8 in the gastric mucosa.
Application of probiotics in the eradication therapy: The clinical application of probiotics in the treatment of H. pylori infection has been performed in many countries for more than 20 years. Now the utilization of probiotics alone for H. pylori infection has almost been settled. Both early and recent reviews[28,29] concluded that probiotics significantly improved gastric mucosal inflammation, and decreased the density of H. pylori on the mucosa. However, to our knowledge, the complete eradication of H. pylori colonizing the stomach by probiotic treatment alone has not been demonstrated. One representative trial of probiotics alone for the treatment of H. pylori infection was reported by Sakamoto et al in 2001. In their study, 31 H. pylori-infected subjects (mean age 50 years) ingested yogurt containing 109 CFU of LG21 or placebo yogurt without LG21 every day for 8 wk. The results of 13C-urea breath tests (UBT) and assays of serum pepsinogens I and II (PGI/II) showed a significant clinical improvement after LG21 yogurt treatment. The 13C-UBT result and the PGI/II ratio are known to indirectly represent H. pylori density and the degree of mucosal inflammation in the stomach, respectively[30,31]. A bacterial examination of gastric mucosal biopsy specimens (by culturing) revealed 2-100-fold decreases in the number of H. pylori. However, there were no subjects in whom H. pylori was completely eliminated. Pantoflickova et al reported the effects of the administration of L. johnsonii La1 (LC-1) to 50 H. pylori-positive healthy volunteers in a randomized controlled, double-blind study. The subjects received 125 g of fermented milk containing 106-107 CFU/g of LC-1 or placebo milk without LC-1 every day for 16 wk. The severity/activity of antral gastritis (assessed histologically) and the H. pylori density (assesses by a 13C-UBT) showed significant improvement. The histological examination of the mucous mucosa also revealed a significant increase in the mucous thickness in the LC-1-treated group. This suggested that the stabilization of the mucosal barrier by probiotics also enhanced the suppression of H. pylori.
Recently, the H. pylori eradication rate in patients treated using anti-microbial agents is decreasing. This is mainly due to antimicrobial resistance. In the early 1990s, the standard triple therapy achieved an eradication rate of > 90%. In contrast in the past decade, the effectiveness of this regimen often falls to < 70%[33,34]. According to an ITT analysis by Deguchi et al in 2012, the successful eradication rate using the same regimen was just 69.3%. In those subjects, the rate of infection with clarithromycin-resistant strains of H. pylori was as high as 27.1%. This increase in resistance to antimicrobials like clarithromycin is thought to have reduced the eradication rate. Actually, the clarithromycin resistance rates of H. pylori isolated from children in North America and Europe were reported to be 10.6%-25% and 1.7%-23.4% respectively[36,37]. These studies also reported the increasing prevalence of H. pylori isolates that are resistant to metronidazole, which is frequently used in the first-line regimen.
The use of probiotics in combination with antimicrobial agents significantly increased the eradication rate, especially for bacteria with antimicrobial resistance. Both the suppressive effect on H. pylori by probiotics and the compliance-promoting effect of ameliorating the side effects of antimicrobials are thought to significantly increase the eradication rate. Actually, Deguchi et al reported that a group treated with one week-triple therapy supplemented with LG21 yogurt and a group with triple therapy alone showed cure rates of 82.6% and 69.3%, respectively. The difference in the intention-to-treat analysis was statistically significant (P = 0.018). In their study, 112 g of yogurt containing 109 CFU of LG21 was given twice daily for 4 wk (3 wk of pretreatment and 1 wk during eradication therapy). According to a recent meta-analysis of 40 eligible studies with 8924 patients, the use of probiotics before and throughout the eradication treatment was associated with a superior eradication effect. Patients who received supplementary probiotics showed a higher eradication rate [relative risk (RR) 1.14, 95%CI: 1.10-1.18, P < 0.001] and lower incidence of total side effects (RR 0.47, 95%CI: 0.39-0.57, P < 0.001) in comparison to the control group without probiotics. In a sub-analysis, Lactobacillus was the best choice among the probiotic strains, and probiotics combined with bismuth quadruple regimen was suggested to be the best combination.
Possible role of probiotics in preventing post-eradication gastric cancer: The lifetime risk of gastric cancers in H. pylori-infected individuals is estimated to be 3%-5%. In H. pylori-infected patients, colonization with H. pylori on the gastric mucosa is known to gradually decrease overtime and often becomes undetectable in patients who develop gastric cancer. Furthermore, during long-term follow-up (up to approximately 20 years) of patients who had been cured of H. pylori infection at the start of observation, 0.35% of subjects developed gastric cancer per year. That is, 7% is estimated to have developed gastric cancer at 20 years. These findings strongly suggest that there are some causative factors other than H. pylori can also promote the development of gastric cancer even after H. pylori eradication. However, H. pylori is currently considered the most important pathogen for the development of gastric cancer.
According to the Correa pathway, chronic H. pylori infection progresses over the decades through the following stages: chronic gastritis, atrophy, intestinal metaplasia, and cancer. Gastric adenocarcinomas are classified as both well-differentiated (intestinal-type) and undifferentiated (diffuse-type) ones. The development of gastric atrophy is recognized as a critical step to the development of intestinal-type gastric cancer in the Correa pathway. Mucosal atrophy is usually accompanied by inflammation, and is thus recognized as atrophic gastritis. Accordingly, atrophic gastritis appears to be the strongest risk factor for gastric cancer. The histological characteristics of the gastric mucosa (e.g., inflammation, atrophy, and intestinal metaplasia) were analyzed to identify risk factors for gastric cancer after H. pylori eradication. The mucosal inflammation score of the group who developed gastric cancer after successful H. pylori eradication (n = 61) was significantly higher than the group without cancer after eradication (n = 122). The RR and 95%CI were 5.92 and 2.11-16.6, respectively (P < 0.01). Neither atrophy nor intestinal metaplasia itself was a direct risk factor for post-eradication cancer.
The gastric corpus and antrum predominantly contain acid-secreting parietal cells and gastrin-secreting G cells, respectively. Thus, the mucosal atrophy in the corpus caused by H. pylori infection rapidly leads to a reduction in the gastric acid production. In contrast, the production of gastrin (an acid secretion stimulating hormone) remains relatively unchanged. Of note, patients who develop H. pylori-associated duodenal ulcers seem to be somewhat protected from the occurrence of gastric cancers. The predominant mechanism of this protection in patients with duodenal ulcer from the cancers appears to be a higher basal level of gastric acid secretion. On the contrary, it was reported that the long-term suppression of gastric acid secretion by proton pump inhibitors (PPIs) was associated with a significantly increased risk of gastric cancer in H. pylori-infected subjects. During approximately 8 years of follow-up, Cheung et al evaluated the gastric cancer risk in patients treated with PPI using a Cox proportional hazards model. The study population consisted of approximately 63000 subjects, who had received clarithromycin-based triple therapy for H. pylori eradication. The use of PPIs was associated with an increased gastric cancer risk (HR 2.44, 95%CI: 1.42-4.20). This result demonstrated that the long-term use of PPIs was still associated with an increased risk of gastric cancer, even after H. pylori eradication. Accordingly, the stomach with low acidity accompanied by gastritis with atrophy and/or intestinal metaplasia must be considered a high-risk environment that predisposes the gastric mucosa to the development of gastric cancer.
Gastric acid reduction invariably results in a marked increase in the number of non-H. pylori bacteria. Due to the low acidity, these bacteria are still viable and show metabolic activity in the stomach. It therefore appears likely that such an enlarged bacterial mass causes the development of gastric cancers even after H. pylori eradication. Recent studies on the characteristics of the gastric microbial changes associated with gastric carcinogenesis revealed a reduction of species richness, the enrichment of intestinal bacteria or an increase of bacterial species of oral cavity origin[47,48]. It seems unlikely that the deoxidization of dietary nitrates to nitrite by such dysbiotic bacteria could rapidly convert dietary amines into carcinogenic N-nitro compounds, because this conversion requires a sufficient amount of acid (which is not present in the stomach with mucosal atrophy).
Sung et al analyzed gastric microbes associated with gastric mucosal inflammation-which is considered to be the strongest risk factor for post-eradication gastric cancer-at one year after H. pylori eradication. They identified several of bacterial groups that were significantly associated with persistent inflammation. These bacteria included the genera Acientobacter, Ralstonia, Actinobacillus and Erwinia, which are all Gram-negative bacteria. Miyata et al isolated several types of Gram-negative bacteria from the H. pylori-infected gastric mucosa, including Fusobacterium, Haemophilus, Neisseria and Veillonella species. Coculture of a gastric epithelial cell line with the lipopolysaccharide (LPS) specimens extracted from these bacterial groups stimulated a significant amount of IL-8 production. Sano et al found high LPS activity in gastric fluid samples with weak acidity (pH > 4), whereas there was little or no activity in those with strong acidity (pH < 2). Spearman’s test demonstrated a close correlation between pH and LPS activity in their 136 samples (r = 0.872) (Figure 3). These findings suggested that LPS from such non-H. pylori Gram-negative bacteria may perpetuate gastric inflammation and accelerate neoplastic progression in the hypochlorhydric stomach after H. pylori eradication.
Figure 3 Correlation between pH and lipopolysaccharide activity in gastric fluid.
The pH values and lipopolysaccharide activity of gastric fluid samples from 136 subjects were examined using a recombinant factor C assay kit. The correlation coefficients of the both parameters by Spearman test (r) is shown on the upper left. LPS: Lipopolysaccharide. Citation: Sano M, Uchida T, Igarashi M, Matsuoka T, Kimura M, Koike J, Fujisawa M, Mizukami H, Monma M, Teramura E, Yoshihara S, Sato H, Morimachi M, Ito A, Ueda T, Shiraishi K, Matsushima M, Suzuki T, Koga Y. Increase in the Lipopolysaccharide Activity and Accumulation of Gram-Negative Bacteria in the Stomach With Low Acidity. Clin Transl Gastroenterol 2020; 11: e00190. Copyright ©Wolters Kluwer Health, Inc. 2020. Published by Wolters Kluwer Health, Inc.
To examine a possible preventive effect of probiotics on post-eradication gastric cancer, we administered a probiotic LG21 strain to subjects with successful eradication who still suffered from atrophic gastritis. In a fasting state in the morning, the pH value and LPS activity of their gastric fluids’ samples were > 3.0 and > 10 EU/mL, respectively (Figure 4). Then, they received 109 CFU of LG21 in yogurt every day for 3 mo. In 8 of 10 subjects, the pH value considerably decreased after LG21 treatment. Lactic acid secreted by the probiotic LG21 strain is thought to restore acidity in the stomach with low acidity. Interestingly, the LPS activity of these subjects, in whom the gastric acidity partially recovered, almost disappeared or markedly decreased. The possible termination of LPS-induced inflammation by LG21 suggests a possible role of probiotics in preventing the development of gastric cancer after H. pylori eradication.
Figure 4 Effect of LG21 administration on the pH and lipopolysaccharide activity in the gastric fluid.
Ten subjects who had gastric fluid (GF) with low acidity and high lipopolysaccharide (LPS) activity consumed yogurt containing 109 CFU of LG21 every day for 3 mo. The pH value and LPS activity in the GF were measured before and after LG21 treatment. LPS: Lipopolysaccharide.