Abstract
Probiotics involving Lactobacillus, Bifidobacterium, Saccharomyces, Clostridium butyricum, etc. have been reported to have inhibitory effects on Helicobacter pylori infections in in vitro and in vivo studies. In addition, these probiotics have been reported to be effective in clinical studies; the patients treated with triple therapy combined with probiotics had a higher H. pylori eradication rate than those with triple therapy only, and antibiotic-associated gastrointestinal side effects during H. pylori eradication therapy were reduced in the patients treated with a probiotics supplemented regimen. It was also reported that probiotic supplementation reduced side effects and permitted a slight improvement in eradicating H. pylori in second-line eradication therapy.
Introduction
Helicobacter pylori has been identified as the etiological agent of chronic active gastritis, peptic ulcer disease Citation[1], Citation[2], gastric adenocarcinoma Citation[3], and mucosal-associated lymphoid tissue (MALT) lymphoma Citation[4]. Triple therapy using two kinds of antibiotics (mainly amoxicillin and clarithromycin) and a proton pump inhibitor has already been established and eradication therapy for H. pylori-infected patients by triple therapy has been reported to be effective. However, the detection rate of drug-resistant H. pylori strains has recently been increasing, and the side effects caused by triple therapy are serious in some patients.
Probiotics have been defined as live microorganisms or microbial supplements of human origin beneficially influencing human health by improving the intestinal microbial balance. Many species of microorganisms are currently available as probiotics (). Probiotics using Lactobacillus, Bifidobacterium, Saccharomyces, Clostridium, etc. have inhibitory effects on H. pylori infection in in vitro and in vivo studies. In the present study, the effect of the probiotic agent, Clostridium butyricum strain MIYAIRI588 (which is available in Japan for treatment of patients with antibiotic-associated diarrhea and Clostridium difficile infection) on H. pylori was examined by in vitro and in vivo experiments.
Table I. Microorganisms used for probiotics.
Materials and methods
H. pylori strain TK1402 was used in the present study. The strain was cultured microaerophilically in Brucella broth containing 10% fetal bovine serum. C. butyricum strain MIYAIRI588 was cultured anaerobically in GAM broth. The harvested culture supernatant of C. butyricum MIYAIRI588 was used in the in vitro experiment to evaluate anti-H. pylori effect with or without pH adjustment to 7.2 by sodium hydroxide. The effect of C. butyricum MIYAIRI588 on adhesion of H. pylori TK1402 to gastric epithelial cells was evaluated by flow cytometry. H. pylori was labeled with lipophilic dye, PKH2. In vivo experiments using germ-free mice (IQI/jic, 8 weeks old, female) were performed to evaluate the effect of C. butyricum MIYAIRI588 on colonization by H. pylori TK1402. Germ-free mice were orally infected three times (once a day) with H. pylori TK1402 (108–9 cfu/0.5 ml). One week after the first inoculation with H. pylori, the gnotobiotic mice were co-infected with C. butyricum MIYAIRI588 (once a day, three times). At 1–5 weeks after inoculation with H. pylori, the gnotobiotic mice were sacrificed, and isolation and identification of H. pylori using Gram stain, urease test, catalase test, and oxidase test were carried out.
Results
We have examined the effect of C. butyricum MIYAIRI 588 on H. pylori in several test models including gnotobiotic mice. The culture supernate of C. butyricum inhibited the growth of H. pylori even when its pH was adjusted to 7.4 (). When 25% of culture supernatant of C. butyricum was used, the inhibitory effect on the growth of H. pylori was not induced. The bactericidal effect of butyric acid on H. pylori was stronger than that of lactic, acetic or hydrochloric acids (data not shown). Flow cytometric analysis showed that pre-incubation of gastric epithelial cells with H. pylori and C. butyricum inhibited the adhesion of H. pylori to the cells (data not shown). In vivo effects of C. butyricum on H. pylori colonization were examined by using germ-free mice. The number of H. pylori in gastric mucosa was significantly reduced by co-infection with vegetative cells of C. butyricum (). Cure of persistent infection with H. pylori in the gnotobiotic mice was demonstrated following infection with spores of C. butyricum.
Table II. Effect of culture supernatants of C. butyricum strain MIYAIRI588 on the growth of H. pylori strain TK1402*.
Table III. Effect of Clostridium butyricum on H. pylori-monoassociated gnotobiotic mice.
Discussion
The published reports Citation[5–12] on the effects of probiotics on H. pylori in in vitro and in vivo studies are listed in . The probiotic microorganisms include Lactobacillus salivarius, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Clostridium butyricum, Bacillus subtilis, and Weissella confusa. These invitro studies indicated that probiotics suppress growth of H. pylori, although the mechanisms for the suppression are not clearly understood. Short chain fatty acid produced by probiotic bacteria might be associated with the inhibitory effect on the growth of H. pylori. However, in the present study it was shown that culture supernatants of C. butyricum MIYAIRI588 with pH adjusted to 7.2 still had an inhibitory effect on the growth of H. pylori.
Table IV. The effects of probiotics on H. pylori infection: in vitro and in vivo studies.
The published reports Citation[13–24] on the effects of probiotics on H. pylori in clinical trials are listed in . Ingestion of L. gasseri strain LG21 did not eradicate H. pylori, but it decreased H. pylori density in gastric mucosa of the H. pylori-positive volunteers Citation[15]. Similar results were reported in clinical trials using L. brevisCitation[21] and yogurt containing L. acidophilus and Bifidobacterium lactisCitation[22]. It was reported that triple therapy supplemented with inactivated culture of L. acidophilusCitation[14] or yogurt containing Lactobacillus and BifidobacteriumCitation[19] increased H. pylori eradication rates. Various side effects such as diarrhea, vomiting, nausea, and taste disturbance are seen during H. pylori eradication therapy. Several reports showed that these side effects were significantly reduced in the patients treated with probiotics-supplemented triple therapy. In the clinical trial reported by Cremonini et al. Citation[18], 85 H. pylori-infected asymptomatic patients were treated with triple therapy combined with Lactobacillus GG, S. boulardii, or Lactobacillus + Bifidobacterium. Side effect frequencies of diarrhea and taste disturbance were significantly lower in the probiotic-treated groups than in the placebo group. In second-line therapy, probiotic supplementation with L. casei was reported to reduce side effects and permit a slight improvement in eradicating H. pyloriCitation[24].
Clinical trials showed that probiotic-supplemented regimen increased eradication rates and reduced side effects during eradication therapy for H. pylori. As probiotics are effective against antibiotic-resistant H. pylori strains, the use of probiotics needs to be considered as a supportive therapy for eradication of H. pylori
Table V. Effect of probiotics on H. pylori infections: clinical trials.
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