A total of 34 patients were enrolled; 31 patients were analyzed in the study. An overview of the study design in shown in Fig. 1. A flow chart describing the patient selection and exclusion is shown in Fig. 2. Demographic data for patients who were analyzed are shown in Table 1.
Table 1
Demographic data of analyzing patients, Mean ± SD
| Variables (Mean ± SD) | 0w | 4w | 8w | 10w | |
| N=31 | |
| Age (years) | 63.7 ± 11.8 | | | | |
| Gender (M/F) | 11/20 | | | | |
| History of gastrointestinal operation, n (%) | 4(14.3%) | | | | |
| Combination use of laxative | | | | | |
| none, n | 4 | | | | |
| Osmotic laxative, n | 18 | | | | |
| Stimulant laxative, n | 11 | | | | |
| lubiprostone/Linaclotide, n | 7 | | | | |
| Kampo medicine, n | 8 | | | | |
| Over-the-counter laxative, n | 3 | | | | |
| Over 2 kinds of laxative, n | 17 | | | | |
| Mean frequency of bowel movements (2 weeks) | 10.2 ± 3.42 | 10.9 ± 3.07 | 11.3 ± 2.77** | 11.1 ± 2.58 | |
| Mean stool consistency (Bristol stool form scale) (2 weeks) | 3.82 ± 1.25 | 3.55 ± 1.22 | 3.98 ± 1.25 | 3.8 ± 1.16 | |
| Mean extent of straining (2 weeks) | 2.96 ± 0.78 | 2.98 ± 0.97 | 2.79 ± 0.97 | 2.85 ± 0.92 | |
| Mean overall PAC-QOL score | 1.73 ± 0.54 | 1.07 ± 0.63** | 0.97 ± 0.65** | 1.1 ± 0.68 | |
| Mean frequency of using rescue laxative (2 weeks) | 1.0 ± 2.1 | 0.81 ± 2.2 | 0.88 ± 2.6 | 0.84 ± 2.7 | |
| The mean proportion of patients who have sense of incomplete evacuation | 71(54.0-87.9) | 58.1(39.7–76.5) | 67.7(50.3–85.2) | 61.3(43.1–79.5) | |
*p < 0.05, **p < 0.01, versus baseline (0w) |
Qol Assessment
The mean overall PAC-QOL score at baseline was 1.73 ± 0.54, whereas the PAC-QOL scores after 4 and 8 weeks of BBG9-1 administration, and after a 2-week washout period, were 1.07 ± 0.63, 0.97 ± 0.65 and 1.1 ± 0.68, respectively. The PAC-QOL score for all patients significantly improved after 4 and 8 weeks of BBG9-1 administration (Fig. 3a. p < 0.05, p < 0.05, respectively). Furthermore, there was no significant difference between the score after 8 weeks of administration and that after the 2-week washout period. Participants in this study showed an equivalent QOL after discontinuance of the probiotic for 2 weeks.
All the subscales score in the PAC-QOL (physical discomfort, worries/concerns, psychosocial discomfort, and satisfaction) were significantly decreased after 4 and 8 weeks of BBG9-1 administration (p < 0.05, p < 0.05, p < 0.05, p < 0.05 respectively) (Fig. 3b-3e).
Stool Consistency Assessment
The mean BSFS score at baseline was 3.82 ± 1.25, whereas the BSFS scores after 4 and 8 weeks of BBG9-1 administration and after a 2-week washout period were 3.55 ± 1.22, 3.98 ± 1.25, and 3.8 ± 1.16, respectively (Fig. 4a). These scores were not significantly different from baselines.
We conducted a post hoc analysis of the BSFS scores based on the scores pre-intervention. For patients who showed had the BSFS score of 4 or more at the start of the study, the mean BSFS score at baseline was 4.71 ± 0.54, whereas the BSFS scores after 4 and 8 weeks of BBG9-1 administration and after a 2-week washout period were 4.35 ± 0.74, 4.5 ± 0.7, and 4.33 ± 0.9, respectively. The BSFS score at 4 weeks of BBG9-1 administration was significantly different from baseline (Fig. 4b, p = 0.03). For patients who had the BSFS score of less than 4 at the start of the study, the mean score at baseline was 2.59 ± 0.83, and the BSFS scores after 4 and 8 weeks of BBG9-1 administration and after a 2-week washout period were 2.43 ± 0.78, 3.27 ± 0.82, and 3.07 ± 1.1, respectively. The BSFS score significantly increased after 8 weeks of BBG9-1 administration (Fig. 4c, p = 0.03).
Frequency Of Bowel Movement
The mean overall frequency of bowel movements at baseline was 10.2 ± 3.42, whereas the frequency of bowel movements after 4 and 8 weeks of BBG9-1 administration and after a 2-week washout period were 10.9 ± 3.07, 11.3 ± 2.77, and 11.1 ± 2.58, respectively. The frequency of bowel movements was significantly increased after 8 weeks of BBG9-1 administration compared to baseline (Fig. 4d, p < 0.01).
For patients who had the BSFS score of 4 or more at the start of the study, the mean overall frequency of bowel movements at baseline was 10.5 ± 3.75, whereas the frequency of bowel movements after 4 and 8 weeks of BBG9-1 administration and after a 2-week washout period were 10.9 ± 3.24, 11.1 ± 3.75, and 11.4 ± 3.02, respectively. None of these were significantly different compared to baseline (Fig. 4h). For patients that had the BSFS score less than 4 at the start of the study, the mean frequency of bowel movements at baseline was 9.9 ± 3.02, whereas the frequency of bowel movements after 4 and 8 weeks of BBG9-1 administration and after a 2-week washout period were 10.8 ± 2.95, 11.5 ± 2.47, and 10.6 ± 2.18, respectively. The frequency of bowel movements significantly increased after 8 weeks of BBG9-1 administration compared to baseline (Fig. 4f, p = 0.04).
Degree Of Straining Assessment
The mean degree of straining at baseline was 2.96 ± 0.78, whereas the degree of straining after 4 and 8 weeks of BBG9-1 administration and after a 2-week washout period were 2.98 ± 0.97, 2.79 ± 0.97, and 2.85 ± 0.92, respectively (Fig. 4g). These values were not significantly different from the baseline. For patients that had the BSFS score of more than 4 at the start of the study, the mean degree of straining at baseline was 2.75 ± 0.73, whereas the mean degrees of straining after 4 and 8 weeks of BBG9-1 administration and after a 2-week washout period were 2.79 ± 0.78, 2.75 ± 1.03, and 2.84 ± 0.89, respectively. These values were not significantly different than baseline (Fig. 4h). However, for patients who had the BSFS score of less than 4 at the start of the study, the mean degree of straining at baseline was 3.25 ± 0.79, whereas the mean degrees of straining after 4 and 8 weeks of BBG9-1 administration and after a 2-week washout period were 3.25 ± 1.16, 2.8 ± 0.9, and 2.87 ± 0.99, respectively. The degree of straining was significantly alleviated after 8 weeks of administration (Fig. 4i, p = 0.03).
Sense Of Incomplete Evacuation Assessment
The mean proportion of patients who had sense of incomplete evacuation was 71% (95% CI, 54.0–87.9), whereas the mean proportion of patients who had a sense of incomplete evacuation after 4 and 8 weeks of BBG9-1 administration and after a 2-week washout period were 58.1% (95% CI, 39.7–76.5), 67.7% (95% CI, 50.3–85.2), and 61.3% (95% CI, 43.1–79.5), respectively (Table 1). These were not significantly different than at baseline.
Frequency Of Rescue Drug Use Assessment
The mean frequency of rescue drug use at baseline was 1.0 ± 2.1, whereas the frequencies of rescue drug use after 4 and 8 weeks of BBG9-1 administration and after a 2-week washout period were 0.81 ± 2.2, 0.88 ± 2.6, and 0.84 ± 2.7, respectively (Table 1). These were not significantly different than at baseline.
In summary, the overall and all the subscales scores in the PAC-QOL significantly improved after BBG9-1 administration. For all patients, there were no significant differences observed comparing before and after BBG9-1 intervention in terms of the BSFS score, degree of straining, sense of incomplete evacuation, and frequency of rescue drug use (Table 1), whereas the frequency of bowel movements was significantly increased after BBG9-1 intervention.
Subset analysis revealed that patients with the initial BSFS score of < 4 had tended to improve to have soft stools, a significant increase in the frequency of their bowel movements, and a significant alleviation in their degree of straining following BBG9-1 administration. Furthermore, patients with the initial BSFS score ≥ 4 tended to improve to have harder stool following BBG9-1 administration.
Analysis of fecal microbiota
For fecal microbiota, there were no significant changes in the Shannon index (evenness) and OTU observed following BBG9-1 treatment (Fig. 5a and 5b). However, chao1 (richness) was significantly increased (Fig. 5c). At the phylum level, Nitrosporae were significantly increased after Bifidobacterium treatment. At the genus level, Sarcina, Johnsonella, Thermodesulfovibrio, Lentibacillus, Yaniella, Marinitoga, Arcanobacterium, Phyllobacterium, Kineosporia, Pseudidiomarina, Ectothiorhodospira, Sphaerisporangium, Rhodobacter, Anaeromusa, and Halanaerobacter were significantly increased, and Neisseria, Leptotrichia, Pasteurella, Abiotrophia, and Achromobacter were significantly decreased after BBG9-1 treatment (Table 2). Additionally, at the species level, Sarcina maxima was significantly increased (Table 3). In the LefSe analysis, the absolute value of the LDA score was 2 or more for Phyllobacteriaceae, Aerococcaceae, Neisseria, Aggregatibactor, Alphaproteobacteria, Haemophillus, and Pasteurellales (Fig. 5e). The cladogram is shown in Fig. 5 f. The functional potential of the bacterial assemblies associated with each stool sample was predicted with PICRUSt using level 3 of the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs. As assessed with LEfSe at a P value < 0.05, the gut microbiome post-treatment was significantly enriched in twenty-six functional categories, compared to pre-treatment. These enriched functional categories (Table 4) were related to carbohydrate metabolism (e.g., propanoate metabolism, butanoate metabolism, starch and sucrose metabolism, and galactose metabolism), amino acid metabolism (e.g., phenylalanine, tyrosine, and tryptophan biosynthesis, tyrosine metabolism, valine, leucine, and isoleucine biosynthesis) and metabolism of cofactors, energy metabolism (methane metabolism) and vitamins (e.g., pantothenate and CoA biosynthesis, retinol metabolism, and lipoic acid metabolism). Surprisingly, propanoate and butanoate metabolism were significantly increased and methane metabolism significantly decreased after BBG9-1 administration.
Table 2
Change of gut-microbiota abundance between pre- and post- Bifidobacterium administration in Genus levels
Genus | Pre (%) | | Post (%) | | p Value | |
Sarcina | 0.042 | | 0.14 | | 0.046 | * |
Neisseria | 0.028 | | 0.015 | | 0.002 | ** |
Johnsonella | 0.058 | | 0.071 | | 0.036 | * |
Thermodesulfovibrio | 0.013 | | 0.019 | | 0.013 | * |
Leptotrichia | 0.0029 | | 0.0017 | | 0.035 | * |
Lentibacillus | 0.0067 | | 0.0089 | | 0.047 | * |
Yaniella | 0.0045 | | 0.0066 | | 0.014 | * |
Marinitoga | 0.0041 | | 0.0058 | | 0.033 | * |
Arcanobacterium | 0.0020 | | 0.0033 | | 0.027 | * |
Phyllobacterium | 0.00064 | | 0.0022 | | 0.009 | ** |
Kineosporia | 0.0020 | | 0.0030 | | 0.037 | * |
Pasteurella | 0.0016 | | 0.00033 | | 0.003 | ** |
Pseudidiomarina | 0.00042 | | 0.0010 | | 0.032 | * |
Abiotrophia | 0.0014 | | 0.000044 | | 0.029 | * |
Ectothiorhodospira | 0.00021 | | 0.00070 | | 0.043 | * |
Sphaerisporangium | 0.00013 | | 0.00057 | | 0.012 | * |
Rhodobacter | 0.00017 | | 0.00059 | | 0.035 | * |
Achromobacter | 0.00033 | | 0 | | 0.012 | * |
Anaeromusa | 0 | | 0.00020 | | 0.044 | * |
Halanaerobacter | 0 | | 0.00020 | | 0.046 | * |
*p < 0.05, **p < 0.01, versus baseline (0w) |
Table 3
Change of gut-microbiota abundance between pre- and post- Bifidobacterium administration in Species levels
Species | Pre (%) | | Post (%) | | p Value |
Anaeromusa acidaminophila | 0 | | 0.00042448 | | 0.043 | * |
Bacteroides rodentium | 0.57 | | 0.31 | | 0.020 | * |
Bacteroides uniformis | 1.5 | | 0.92 | | 0.044 | * |
Bifidobacterium ruminantium | 0.008 | | 0.013 | | 0.020 | * |
Blautia hydrogenotrophica | 0.12 | | 0.24 | | 0.024 | * |
Campylobacter faecalis | 0 | | 0.00042 | | 0.043 | * |
Cohnella laeviribosi | 0.0025 | | 0.0049 | | 0.027 | * |
Ectothiorhodospira imhoffii | 0.00042 | | 0.00127 | | 0.030 | * |
Euzebya tangerina | 0.0072 | | 0.0099 | | 0.031 | * |
Ferrimicrobium acidiphilum | 0.00032 | | 0.00117 | | 0.043 | * |
Fructobacillus pseudoficulneus | 0.0031 | | 0.0060 | | 0.044 | * |
Fusobacterium nucleatum | 0.0023 | | 0.00032 | | 0.012 | * |
Fusobacterium periodonticum | 0.0129 | | 0.0041 | | 0.045 | * |
Halanaerobacter chitinivorans | 0 | | 0.00042 | | 0.043 | * |
Johnsonella ignava | 0.11 | | 0.13 | | 0.044 | * |
Kineosporia mikuniensis | 0.0035 | | 0.0056 | | 0.028 | * |
Lactobacillus pobuzihii | 0.00042 | | 0.00000 | | 0.043 | * |
Leuconostoc carnosum | 0 | | 0.0011 | | 0.048 | * |
Marinitoga okinawensis | 0.00021 | | 0.0011 | | 0.018 | * |
Mycobacterium lepromatosis | 0.00053 | | 0.00011 | | 0.043 | * |
Neisseria lactamica | 0.0011 | | 0.00011 | | 0.048 | * |
Neisseria mucosa | 0.041 | | 0.027 | | 0.001 | ** |
Nocardia devorans | 0.00042 | | 0 | | 0.043 | * |
Paenibacillus filicis | 0.00011 | | 0.00053 | | 0.043 | * |
Pasteurella pneumotropica | 0.0024 | | 0.00064 | | 0.007 | |
Porphyromonas canis | 0.0087 | | 0.014 | | 0.038 | * |
Prevotella enoeca | 0.00042 | | 0 | | 0.043 | * |
Prevotella veroralis | 0.00084 | | 0.0030 | | 0.001 | ** |
Sarcina maxima | 0.080 | | 0.26 | | 0.046 | * |
Sphaerisporangium rubeum | 0.00021 | | 0.0011 | | 0.009 | ** |
Streptococcus salivarius | 0.00011 | | 0.0014 | | 0.021 | * |
Streptomyces roseogilvus | 0.00021 | | 0.0011 | | 0.043 | * |
Syntrophomonas bryantii | 0 | | 0.00042 | | 0.043 | * |
Tepidanaerobacter syntrophicus | 0.011 | | 0.016 | | 0.049 | * |
Thermodesulfovibrio thiophilus | 0.026 | | 0.035 | | 0.040 | * |
Veillonella denticariosi | 0.014 | | 0.006 | | 0.009 | ** |
Vibrio porteresiae | 0.0016 | | 0.0040 | | 0.037 | * |
*p < 0.05, **p < 0.01, versus baseline (0w) |
Table 4
Functional profile of the gut-microbiota from pre- or post- treatment by Bifidobacterium
Level 1 | Level 2 | Level 3 | Pre (N = 30) | | Post (N = 30) | | P value | |
Metabolism | Metabolism of Terpenoids and Polyketides | Limonene and pinene degradation | 0.080 | | 0.088 | | 0.005 | ** |
Metabolism | Carbohydrate Metabolism | Propanoate metabolism | 0.509 | | 0.523 | | 0.007 | ** |
Metabolism | Amino Acid Metabolism | Phenylalanine, tyrosine and tryptophan biosynthesis | 0.889 | | 0.866 | | 0.013 | ** |
Metabolism | Carbohydrate Metabolism | Butanoate metabolism | 0.583 | | 0.607 | | 0.013 | * |
Metabolism | Carbohydrate Metabolism | Starch and sucrose metabolism | 1.139 | | 1.114 | | 0.014 | * |
Metabolism | Metabolism of Cofactors and Vitamins | Pantothenate and CoA biosynthesis | 0.644 | | 0.632 | | 0.018 | * |
Metabolism | Xenobiotics Biodegradation and Metabolism | Drug metabolism - cytochrome P450 | 0.036 | | 0.041 | | 0.024 | * |
Metabolism | Energy Metabolism | Methane metabolism | 1.413 | | 1.375 | | 0.027 | * |
Metabolism | Xenobiotics Biodegradation and Metabolism | Benzoate degradation | 0.209 | | 0.226 | | 0.028 | * |
Metabolism | Xenobiotics Biodegradation and Metabolism | Metabolism of xenobiotics by cytochrome P450 | 0.034 | | 0.040 | | 0.028 | * |
Metabolism | Amino Acid Metabolism | Tyrosine metabolism | 0.352 | | 0.363 | | 0.028 | * |
Metabolism | Metabolism of Cofactors and Vitamins | Retinol metabolism | 0.032 | | 0.035 | | 0.032 | * |
Metabolism | Metabolism of Terpenoids and Polyketides | Biosynthesis of vancomycin group antibiotics | 0.061 | | 0.058 | | 0.033 | * |
Human Diseases | Infectious Diseases | Epithelial cell signaling in Helicobacter pylori infection | 0.098 | | 0.095 | | 0.037 | * |
Organismal Systems | Nervous System | Glutamatergic synapse | 0.110 | | 0.107 | | 0.037 | * |
Metabolism | Xenobiotics Biodegradation and Metabolism | Aminobenzoate degradation | 0.103 | | 0.112 | | 0.040 | * |
Metabolism | Carbohydrate Metabolism | Galactose metabolism | 0.860 | | 0.842 | | 0.042 | * |
Unclassified | Metabolism | Nucleotide metabolism | 0.037 | | 0.046 | | 0.042 | * |
Human Diseases | Neurodegenerative Diseases | Amyotrophic lateral sclerosis (ALS) | 0.008 | | 0.011 | | 0.045 | * |
Metabolism | Metabolism of Terpenoids and Polyketides | Polyketide sugar unit biosynthesis | 0.214 | | 0.206 | | 0.045 | * |
Metabolism | Lipid Metabolism | Synthesis and degradation of ketone bodies | 0.021 | | 0.024 | | 0.045 | * |
Metabolism | Metabolism of Cofactors and Vitamins | Lipoic acid metabolism | 0.021 | | 0.024 | | 0.047 | * |
Metabolism | Xenobiotics Biodegradation and Metabolism | Dioxin degradation | 0.079 | | 0.084 | | 0.047 | * |
Human Diseases | Neurodegenerative Diseases | Huntington's disease | 0.016 | | 0.019 | | 0.049 | * |
Metabolism | Xenobiotics Biodegradation and Metabolism | Caprolactam degradation | 0.021 | | 0.029 | | 0.050 | * |
Metabolism | Amino Acid Metabolism | Valine, leucine and isoleucine biosynthesis | 0.831 | | 0.816 | | 0.0498 | * |
*p < 0.05, **p < 0.01, versus baseline (0w) |
All participants showed over 80% medication compliance. No adverse events were observed during the study period.