Discussion
A randomized, double-blind, placebo-controlled clinical trial of IBS was done for 4 weeks. Compared with placebo, multispecies probiotics were effective for global relief of IBS symptoms as well as for various secondary end-points (i.e. abdominal pain/discomfort and bloating). In addition, probiotics and placebo had different effects on the composition of fecal microbiota.
Multispecies probiotics was used for the treatment of IBS in our study: three Bifidobacterium species, two Lactobacillus species, and one Streptococcus species. The reason is as follows. The level of Bifidobacteria and Lactobacilli species was lower in IBS patients compared with healthy persons.[13,14] Also, S. thermophillus showed the reduction of tumor necrosis factor-alpha caused by lipopolysaccharide in the intestinal barrier.[15] Then, several studies showed that the supplement of Lactobacillus, Bifidobacterium species, or mixtures including species of the genera was effective in alleviating symptoms of IBS.[16]
In this study, the multispecies probiotics were more effective than the placebo group in terms of the primary efficacy end-point. Secondary end-points were achieved in the probiotics group but not placebo group. This finding is consistent with previous data from multispecies probiotics treatment of IBS.[17,18,19] Multispecies probiotics may have a variety of different beneficial effects on IBS symptoms because each species act in a particular way on the gastrointestinal tract, and two or more species acting together may have a synergistic effect. However, the changes in stool frequency and consistency in the probiotics group was similar to those in the placebo group. This may be because the patients had three different subtypes of IBS (IBS with diarrhea, IBS with constipation, and mixed-type IBS) rather than a single subtype. The changes of IBS symptoms relative to baseline were not significantly greater in the probiotics group compared with the placebo group. Although the change of abdominal pain was more improved in the probiotics group, it did not reach the statistical significance (−37% vs −9.2%, P = 0.07). This result seems to be caused by the relatively low number of subjects in this study (the sample size of each arm was 25 patients) because the calculation of sample size was performed based on the primary end-point not the secondary end-points.
To investigate the alterations in intestinal microbiota, fecal microflora was analyzed in this study. Interestingly, numbers of B. lactis, L. rhamnosus, and S. thermophilus increased after week 4 in the probiotics group, whereas only the number of B. lactis increased in the placebo group. In other words, only three of the species in the probiotics mixture remained in the gut after 4 weeks even though there were six species in the mixture. Our findings differ from previous observations. Kajander et al. reported that Bifidobacterium species decreased after treatment with a probiotic mixture of L. rhamnosus, B. breve, and Propinibacterium freudenreichii.[20] Firmesse et al. reported no difference in the composition of gut microbiota after treatment with L. rhamnosus.[21] We found no significant change in the E. coli subgroup, C. perfringens, or the Bacteroides group after treatment, whereas Lyra et al. reported elevated levels of C. thermosuccinogenes following multispecies probiotics treatment that included Bifidobacterium and Lactobacillus species.[22]
Based on the changes in fecal microbiota after probiotics, we thought the relationship between the alterations in gut microflora and the alleviation of IBS symptoms. There is some difficulty in analyzing the direct correlation between IBS symptoms and the alterations of gut microflora because the total number of subjects in fecal analysis (n = 34) was smaller than that of subjects with probiotics or placebo treatment (n = 49). In spite of this discrepancy, we cautiously assumed that the alleviations of IBS symptoms in probiotics groups after 4 weeks were probably associated with significant increases in B. lactis, L. rhamnosus, and S. thermophilus. Interestingly, compared with baseline, the counts of B. lactis at week 4 were increased in the placebo group, although placebo did not show the improvement of IBS symptoms. Maybe, the diet induced the alteration of B. lactis in the placebo group. Despite the drugs that affect intestinal microbiota (e.g. probiotics, prebiotics, synbiotics and antibiotics) were equally restricted in both probiotics and placebo groups, it is very difficult to control the dietary habits in all participants during 4 weeks. So, it seemed that the efficacy of probiotics on IBS symptoms may be associated with the synergistic effect of three strains (B. lactis, L. rhamnosus, and S. thermophilus ) rather than that of single strain (B. lactis ). Also, we can consider the metabolic effect of probiotic strains on gastrointestinal tract. Several studies demonstrated that the probiotic supplement including Lactobacilli and Streptococcus species induced the increase of short-chain fatty acids (SCFAs) in colon lumen and then decreased fecal pH.[23,24] The change of SCFA and fecal pH are considered to contribute the improvement of gut motility. We assumed that L. rhamnosus and S. thermophilus in the probiotics group could improve IBS symptoms by the change of SCFA and fecal pH.
To identify the alterations of gut microbiota, quantitative reverse transcription–PCR (qRT-PCR) was used in our study. Among the commonly used molecular techniques, for example denaturing gradient gel electrophoresis (DGGE) or temperature gradient gel electrophoresis, terminal-restriction fragment length polymorphism, and dot blot hybridization, qRT-PCR provides a rapid, precise quantification of the genus or species, and has been applied to human feeding studies.[25,26,27] Recently, new approaches to gut microbiota have been tried other than commonly used method such as 16S rDNA. So called metagenomic sequencing can reveal the combined genomes of the gut microflora, non-cultured ones, and functional dysbiosis beyond compositional dysbiosis. In spite of these merits, however, it is not widely used, and there is no information on microbial expressed functions.[27] The global changes of intestinal mirobiota can provide us with a role of the probiotics in IBS. Determining the global changes of fecal microflora after probiotics supplement was limited because complete sequencing was not done. There is a need to perform in-depth methods including a complete sequencing for intestinal microflora after probiotic supplement in future studies.
Several reports demonstrated that IBS patients had more temporal instability of fecal microbiota than healthy controls.[28,29] In this study, concordance of PCR-DGGE using fecal DNAs from each group was done to evaluate the compositional change in the fecal microbiota between before and after treatment. PCR-DGGE was done in some patients, but not all. The placebo group showed a lower concordance rate of DGGE profiles than the probiotics group between before and after treatment, but it did not reach the significant difference statistically (P = 0.086). Although all the fecal samples of each group were not analyzed, the result indicates that the test product contributed to the maintenance of the compositional stability of the intestinal microbiota. The clinical improvements in this study may be associated with the maintenance of the compositional stability of the intestinal microbiota.
Our study has some limitations. First, fecal microflora were analyzed in only 75.6% of the patients (34/49) because we only analyzed stool samples from those who consented. As mentioned earlier, it was not easy to assess a direct relationship between alterations in gut microbiota and improvements in IBS symptoms in patients who have taken probiotics supplements. Even though the present study has this weak point, the probiotics group showed alleviations of IBS symptoms such as abdominal pain and bloating with increases in the counts of B. lactis, L. rhamnosus, and S. thermophilus. Second, we evaluated the intestinal microbiota by fecal microflora analysis. There are two methods for analyzing gut microbiota: fecal microflora analysis reflects the composition of the luminal intestinal microbiota, while culture of intestinal tissue reflects that of the mucosal-associated intestinal microbiota. Parkes et al. reported that luminal microbiota were associated with gas production through carbohydrate fermentation, whereas mucosal-associated microbiota might play a role in immune responses to microbes.[30] Despite these theoretical differences, the luminal and mucosal-associated intestinal microbiota in IBS patients were found to be similar.[31] Third, a validated quality of life was not measured in this study, although we checked a global relief of IBS symptoms after treatment. Several reports indicated that probiotics improved quality of life in patients with IBS.[32,33] Our study focused on the improvement of IBS symptoms and gut microbiota alterations after probiotic supplement. Fourth, we did not perform a separate analysis of therapeutic effect on IBS according to gender or IBS subtypes. The reason is as follows. There were a small number of subjects present in some subgroup (e.g. the number of women in placebo group was six), although baseline characteristics of the participants were not statistically different between the probiotic and placebo group ( Table 2 ). In case of the small sample sizes in subgroup analyses, the statistical power is not enough because of increase in type I error.[34]
In conclusion, multispecies probiotics given to IBS patients are effective in the global relief of IBS symptoms as well as in alleviating abdominal pain, discomfort and bloating. Furthermore, the multispecies probiotics induced the alterations of intestinal microbiota. These findings support that probiotics therapy is effective by mechanism of gut microbiota alterations in IBS.
J Gastroenterol Hepatol. 2014;29(1):52-59. © 2014 Blackwell Publishing
