Lactobacillus paracasei subsp. paracasei 2004 improves health and lifespan in Caenorhabditis elegans.

Recent research has highlighted the importance of the gut microbiome in regulating aging, and probiotics are interventions that can promote gut health. In this study, we surveyed several novel lactic acid bacteria to examine their beneficial effect on organismal health and lifespan in C. elegans. We found that animals fed some lactic acid bacteria, including L. acidophilus 1244 and L. paracasei subsp. paracasei 2004, grew healthy. Supplementation with the lactic acid bacterial strains L. acidophilus 1244 or L. paracasei subsp. paracasei 2004 significantly improved health, including food consumption, motility, and resistance to oxidative stressor, hydrogen peroxide. Our RNA-seq analysis showed that supplementation with L. paracasei subsp. paracasei 2004 significantly increased the expression of daf-16, a C. elegans FoxO homolog, as well as genes related to the stress response. Furthermore, daf-16 deletion inhibited the longevity effect of L. paracasei subsp. paracasei 2004 supplementation. Our results suggest that L. paracasei subsp. paracasei 2004 improves health and lifespan in a DAF-16-dependent manner.

Effects of Bifidobacterium longum CLA8013 on bowel movement improvement: a placebo-controlled, randomized, double-blind study.

A placebo-controlled, randomized, double-blind study was conducted to evaluate the effect of taking 25 billion colony-forming units of heat-killed Bifidobacterium longum CLA8013 over 2 weeks on bowel movements in constipation-prone healthy individuals. The primary endpoint was the change in defecation frequency between the baseline and 2 weeks after the intake of B. longum CLA8013. The secondary endpoints were the number of days of defecation, stool volume, stool consistency, straining during defecation, pain during defecation, feeling of incomplete evacuation after defecation, abdominal bloating, fecal water content, and the Japanese version of the Patient Assessment of Constipation Quality of Life. A total of 120 individuals were assigned to two groups, 104 (control group, n=51; treatment group, n=53) of whom were included in the analysis. After 2 weeks of consuming the heat-killed B. longum CLA8013, defecation frequency increased significantly in the treatment group compared with that in the control group. Furthermore, compared with the control group, the treatment group showed a significant increase in stool volume and significant improvement in stool consistency, straining during defecation, and pain during defecation. No adverse events attributable to the heat-killed B. longum CLA8013 were observed during the study period. This study revealed that heat-killed B. longum CLA8013 improved the bowel movements of constipation-prone healthy individuals and confirmed that there were no relevant safety issues.

Relationship between Chemotherapy-Induced Diarrhea and Intestinal Microbiome Composition.

BACKGROUND AND AIM: Fluoropyrimidines (FPs) are key drugs in many chemotherapy regimens; however, recipients are often prone to diarrhea due to gastrointestinal toxicity. Disruption of the intestinal epithelial barrier function by FPs leads to dysbiosis, which may exacerbate intestinal epithelial cell damage as a secondary effect and trigger diarrhea. However, despite studies on chemotherapy-induced changes in the intestinal microbiome of humans, the relationship between dysbiosis and diarrhea is unclear. In this study, we aimed to investigate the relationship between chemotherapy-induced diarrhea and the intestinal microbiome. METHODS: We conducted a single-center prospective observational study. Twenty-three patients who received chemotherapy, including FPs as first-line chemotherapy for colorectal cancer, were included. Stool samples were collected before the start of chemotherapy and after one cycle of treatment to analyze intestinal microbiome composition and perform PICRUSt predictive metagenomic analysis. RESULTS: Gastrointestinal toxicity was observed in 7 of 23 patients (30.4%), diarrhea was observed in 4 (17.4%), and nausea and anorexia were observed in 3 (13.0%). In 19 patients treated with oral FPs, the α diversity of the microbial community decreased significantly following chemotherapy only in the diarrheal group. At the phylum level, the diarrheal group showed a significant decrease in the abundance of Firmicutes and a significant increase in the abundance of Bacteroidetes with chemotherapy (p = 0.013 and 0.011, respectively). In the same groups, at the genus level, Bifidobacterium abundance was significantly decreased (p = 0.019). In contrast, in the non-diarrheal group, Actinobacteria abundance increased significantly with chemotherapy at the phylum level (p = 0.011). Further, Bifidobacterium, Fusicatenibacter, and Dorea abundance significantly increased at the genus level (p = 0.006, 0.019, and 0.011, respectively). The PICRUSt predictive metagenomic analysis revealed that chemotherapy caused significant differences in membrane transport in KEGG pathway level 2 and in 8 KEGG pathway level 3, including transporters and oxidative phosphorylation in the diarrhea group. CONCLUSION: Organic-acid-producing bacteria seem to be involved in diarrhea associated with chemotherapy, including FPs.

Diarrhea-predominant Irritable Bowel Syndrome-like Symptoms in Patients With Quiescent Crohn's Disease: Comprehensive Analysis of Clinical Features and Intestinal Environment Including the Gut Microbiome, Organic Acids, and Intestinal Permeability.

Background/Aims: Diarrhea-predominant irritable bowel syndrome (IBS-D)-like symptoms frequently occur in patients with quiescent Crohn's disease (CD). To investigate the factors underlying IBS-D-like symptoms in patients with quiescent CD, we performed a comprehensive analysis of the clinical features and intestinal environment in those patients. Methods: We performed a prospective observational study of 27 patients with quiescent CD (CD activity index [CDAI] ≤ 150; C-reactive protein ≤ 0.3 mg/dL). The presence and severity of IBS-D-like symptoms, health-related quality of life, disease-specific quality of life, and status of depression and anxiety were evaluated. The level of intestinal permeability, fecal calprotectin and organic acids and the profiles of gut microbiome were analyzed. Results: Twelve of the 27 patients with quiescent CD (44.4%) had IBS-like symptoms, and these patients showed a significantly higher CDAI, IBS severity index and anxiety score than those without. The inflammatory bowel disease questionnaire score was significantly lower in the patients with IBS-D-like symptoms. There were no significant differences in small intestinal/colonic permeability or the levels of organic acids between the patients with and without IBS-D-like symptoms. Fusicatenibacter was significantly less abundant in the patients with IBS-D-like symptoms whereas their fecal calprotectin level was significantly higher (384.8 ± 310.6 mg/kg) than in patients without (161.0 ± 251.0 mg/kg). The receiver operating characteristic curve constructed to predict IBS-D-like symptoms in patients with quiescent CD using the fecal calprotectin level (cutoff, 125 mg/kg) showed a sensitivity and specificity of 73.3% and 91.7%, respectively. Conclusion: Minimal inflammation is closely associated with the development of IBS-D-like symptoms in patients with quiescent CD.

Alterations in the gut microbiome in patients with esophageal carcinoma in response to esophagectomy and neoadjuvant treatment.

PURPOSE: Analyzing the gut microbiome is essential for planning treatment strategies to manage esophageal squamous cell carcinoma. This study aimed to characterize the gut microbiome of patients with esophageal squamous cell carcinoma and to identify alterations in its composition during treatment. METHODS: We observed alterations in the gut microbiome in 21 consecutive patients with esophageal squamous cell carcinoma at five different time points, from neoadjuvant treatment to postoperative surgery. Ten healthy individuals were used as a non-cancer control group. Fecal samples were collected and analyzed using 16S ribosomal ribonucleic acid sequencing. RESULTS: Before treatment, participants with esophageal squamous cell carcinoma had different alpha and beta diversity in comparison to healthy controls. The number of Streptococcus, a facultative anaerobic bacterium, was significantly higher, whereas that of Faecalibacterium, an obligate anaerobic bacterium, was significantly lower. Both alpha and beta diversity remained unchanged during neoadjuvant treatment, but the alterations were pronounced after surgery. The increase in the relative abundance of Streptococcus and the decrease in that of Faecalibacterium also tended to be more pronounced after surgery. CONCLUSIONS: The gut microbiome in patients with esophageal squamous cell carcinoma is altered with surgical intervention.

Correction: Improvement of loperamide-induced slow transit constipation by Bifidobacterium bifidum G9-1 is mediated by the correction of butyrate production and neurotransmitter profile due to improvement in dysbiosis.

[This corrects the article DOI: 10.1371/journal.pone.0248584.].

Effect of a High-Fat Diet on the Small-Intestinal Environment and Mucosal Integrity in the Gut-Liver Axis.

Although high-fat diet (HFD)-related dysbiosis is involved in the development of steatohepatitis, its pathophysiology especially in the small intestine remains unclear. We comprehensively investigated not only the liver pathology but also the microbiome profile, mucosal integrity and luminal environment in the small intestine of mice with HFD-induced obesity. C57BL/6J mice were fed either a normal diet or an HFD, and their small-intestinal contents were subjected to microbial 16S rDNA analysis. Intestinal mucosal permeability was evaluated by FITC-dextran assay. The levels of bile acids in the small-intestinal contents were measured by liquid chromatography/mass spectrometry. The expression of tight junction molecules, antimicrobial peptides, lipopolysaccharide and macrophage marker F4/80 in the small intestine and/or liver was examined by real-time RT-PCR and immunohistochemistry. The abundance of Lactobacillus was markedly increased and that of Clostridium was drastically decreased in the small intestine of mice fed the HFD. The level of conjugated taurocholic acid was significantly increased and those of deconjugated cholic acid/secondary bile acids were conversely decreased in the small-intestinal contents. The expression of occludin, antimicrobial Reg IIIß/γ and IL-22 was significantly decreased in the small intestine of HFD-fed mice, and the intestinal permeability was significantly accelerated. Infiltration of lipopolysaccharide was significantly increased in not only the small-intestinal mucosa but also the liver of HFD-fed mice, and fat drops were apparently accumulated in the liver. Pathophysiological alteration of the luminal environment in the small intestine resulting from a HFD is closely associated with minimal inflammation involving the gut-liver axis through disturbance of small-intestinal mucosal integrity.

Probiotic Bifidobacterium bifidum G9-1 Has a Preventive Effect on the Acceleration of Colonic Permeability and M1 Macrophage Population in Maternally Separated Rats.

Although probiotics may be useful for the treatment of irritable bowel syndrome (IBS), it is unclear how probiotics play a role in colonic mucosal integrity and immunity. Here, we aimed to investigate the effect of Bifidobacterium bifidum G9-1 (BBG9-1) on colonic mucosal integrity and macrophage behavior in rats subjected to maternal separation (MS) as a model of IBS. MS pups were individually separated from their mother rats, and a proportion of the MS rats were orally administered BBG9-1. The colonic mucosal permeability was evaluated by Ussing chamber assay. The expression of tight junction proteins and cytokines and the population of CD80-positive cells was examined in the colonic tissues by real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Caco2 cells were stimulated with cytokines and the transepithelial electric resistance (TEER) was measured. MS rats showed significantly higher colonic permeability and lower claudin 4 expression in the colonic epithelium relative to controls. The number of CD80-positive macrophages was significantly increased in the colonic mucosa of MS rats, accompanied by the increase of IL-6 and IFN-γ expression. BBG9-1 treatment ameliorated the increase of M1 macrophage and IL-6/IFN-γ expression in the colonic tissue of MS rats. Simultaneously, BBG9-1 treatment improved the enhanced mucosal permeability and the decreased claudin 4 expression in the colon of MS rats. IL-6 and IFN-γ, whose expression is enhanced in the colon of MS rats, significantly decreased TEER in Caco2 cells in vitro. Probiotic BBG9-1 has a preventive effect on the acceleration of colonic permeability and M1 macrophage population in maternally separated rats.

Improvement of loperamide-induced slow transit constipation by Bifidobacterium bifidum G9-1 is mediated by the correction of butyrate production and neurotransmitter profile due to improvement in dysbiosis.

A treatment option for constipation that improves the quality of life is needed since available laxatives do not effectively improve the quality of life in patients with constipation. A significant association between gut dysbiosis and constipation is recognized, suggesting that probiotics may be an important option for management of constipation. The underlying mechanism by which probiotics improve constipation remains unclear. In this study, we aimed to evaluate the effects of the probiotic Bifidobacterium bifidum G9-1 (BBG9-1) on loperamide-induced delayed colonic transit constipation and to elucidate its mechanism of action. First, the effect of BBG9-1 was evaluated in a rat model of constipation induced by subcutaneous administration of loperamide. BBG9-1 improved constipation parameters (number of feces, fecal water content, and fecal hardness) in constipated rats. Next, the relationship of organic acids and neurotransmitters to gut microbiota was investigated. BBG9-1 improved dysbiosis and prevented a decrease in butyric acid concentration in the gut, increased serum serotonin, and suppressed an increase in dopamine and a decrease in acetylcholine in serum. Further, an increase in the expression level of tryptophan hydroxylase 1, a 5-HT-synthetizing enzyme, was observed. These results suggest that BBG9-1 improves dysbiosis, which results in an increase in organic acids and improvement of neurotransmission. These actions may increase intestinal mobility, finally leading to alleviating constipation. The probiotic BBG9-1 may, therefore, be a potential option for the treatment of constipation.

Bifidobacterium bifidum G9-1 ameliorates soft feces induced by metformin without affecting its antihyperglycemic action.

Recent studies of metformin, the first-line drug for type 2 diabetes, have reported the involvement of gut microbiota in the mechanism underlying its antihyperglycemic effect. However, the mechanisms underlying the development of diarrhea and bloating, which are adverse effects of metformin, are unclear, and these effects decrease the quality of life of metformin-receiving patients with diabetes. In this study, we focused on the effects of metformin on gut microbiota. Namely, we examined the effects of Bifidobacterium bifidum G9-1 (BBG9-1), which has the ability to improve dysbiosis, on the changes in gut microbiota and occurrence of soft feces (increased fecal water content) during the administration of metformin. The results showed that coadministration of BBG9-1 and metformin suppressed metformin-mediated changes in the gut microbiota and, thus, soft feces. Meanwhile, BBG9-1 did not influence the antihyperglycemic effect of metformin. Based on these results, we believe that BBG9-1, which could improve gut microbiota, suppresses metformin-induced soft feces without influencing the drug's antihyperglycemic effect.

Alteration of Colonic Mucosal Permeability during Antibiotic-Induced Dysbiosis.

Although dysbiosis is likely to disturb the mucosal barrier system, the mechanism involved has remained unclear. Here, we investigated alterations of colonic mucosal permeability and tight junction (TJ) molecules in mice with antibiotic-induced dysbiosis. Mice were orally administered vancomycin or polymyxin B for 7 days, and then fecal samples were subjected to microbial 16S rRNA analysis. The colonic mucosal permeability was evaluated by chamber assay. The colonic expression of TJ molecules and cytokines was examined by real-time RT-PCR, Western blotting, and immunohistochemistry. Caco2 cells were stimulated with cytokines and their transepithelial electric resistance (TEER) was measured. Vancomycin-treated mice showed significantly lower gut microbiota diversity than controls, and the same tendency was evident in polymyxin B-treated mice. The colonic mucosal permeability was significantly elevated in both vancomycin- and polymyxin B-treated mice. The expression of claudin 4 in the colonic mucosa was decreased in both vancomycin- and polymyxin B-treated mice. Colonic expression of TNF-α and/or IFN-γ was significantly increased in mice that had been administered antibiotics. TNF-α and IFN-γ stimulation dose-dependently decreased TEER in Caco2 cells. Antibiotic-induced dysbiosis is correlated with the enhancement in colonic tissue permeability, accompanied by a reduction in claudin 4 expression and enhancement in TNF-α and/or IFN-γ expression in mice.

Probiotic Bifidobacterium bifidum G9-1 ameliorates phytohemagglutinin-induced diarrhea caused by intestinal dysbiosis.

Diarrhea is largely caused by dysbiosis accompanying the hyperproliferation of Escherichia coli (E. coli). While current treatments can resolve the symptoms, they cannot suppress the proliferation of pathogenic bacteria in the intestine. Probiotics have numerous beneficial effects on host health, including restoring the balance of the intestinal microbiota. This study investigated the effect of the probiotic Bifidobacterium bifidum G9-1 (BBG9-1), which is active in intestinal dysbiosis, in the incidence of diarrhea, in the composition of the intestinal microbiota, and in the intestinal tissue of a rat model of phytohemagglutinin (PHA)-induced diarrhea. The rats were treated with PHA, with and without BBG9-1, and the microbiota composition throughout the intestine and stool was examined using high-throughput 16S rRNA sequencing. In line with previous reports, PHA administration caused diarrhea as well as dysbiosis due to E. coli hyperproliferation. Histological findings indicated that the jejunal villus length was shortened. Rats that received BBG9-1 showed clear improvements in dysbiosis, diarrhea symptoms, and jejunal villus length. Principal coordinates analysis demonstrated the microbiota profile to be more similar between the BBG9-1 and normal groups than between the PHA and normal groups. These results indicated that BBG9-1 suppresses the hyperproliferation of E. coli and restores the jejunal villus length, thereby improving dysbiosis, and in turn, alleviating the symptoms of diarrhea.

Alleviation of low-fiber diet-induced constipation by probiotic Bifidobacterium bifidum G9-1 is based on correction of gut microbiota dysbiosis.

Constipation, a functional disorder of the digestive system, is common in children and adults and may compromise patient quality of life. Because many patients are not satisfied with the efficacy of existing therapies, in this study, we investigated the efficacy of the probiotic Bifidobacterium bifidum G9-1 (BBG9-1) in constipation induced by a low-fiber diet. After inducing constipation in rats by feeding a low-fiber diet, rats were fed a low-fiber diet mixed with BBG9-1 in 14 days to determine the efficacy of BBG9-1 for alleviating constipation. BBG9-1 significantly alleviated the dysbiosis induced by a low-fiber diet and improved the fecal counts, fecal weights, and fecal water contents. Moreover, it also improved organic acid concentrations in the cecal contents. These results suggested that in low-fiber diet-induced constipation, BBG9-1 could alleviate dysbiosis and constipation and may improve the intestinal environment, supporting its potential application in the treatment of constipation.

Effect of probiotic Bifidobacterium bifidum G9-1 on the relationship between gut microbiota profile and stress sensitivity in maternally separated rats.

Although gut microbiota and early life events are likely involved in the development of irritable bowel syndrome (IBS), it remains unclear how these factors interact in the pathophysiology of IBS. In the present study, using rats subjected to maternal separation (MS) as a model of IBS, we investigated interrelationships among gut microbiota, stress susceptibility and intestinal permeability, and examined the effect of the probiotic Bifidobacterium bifidum G9-1 (BBG9-1) on those interrelationships. When compared with the controls at postnatal day 20, MS rats showed hypercorticosteronemia, enhanced intestinal permeability and changes in gut microbiota structure. All of these changes in MS rats were prevented by treatment with BBG9-1. Although the gut microbiota profile and basal serum corticosterone level did not differ between MS and control rats at postnatal day 56, MS rats showed hypersensitivity to restraint stress in terms of serum corticosterone level and fecal frequency. However, such hypersensitivity was not observed in MS rats treated with BBG9-1. These findings suggest that MS initiates the link between gut microbiota alteration and hypersensitivity to stress and that the triggering of this process can be prevented by the treatment with the probiotic BBG9-1.

Oral administration of Bifidobacterium bifidum G9-1 alleviates rotavirus gastroenteritis through regulation of intestinal homeostasis by inducing mucosal protective factors.

Human rotavirus (RV) infection is a leading cause of dehydrating diarrhea in infants and young children worldwide. Since therapeutic approaches to RV gastroenteritis are limited to alleviation of dehydration with oral rehydration solutions, more direct approaches to palliate symptoms of RV gastroenteritis are required. Treatments with probiotics have been increasingly recognized as alternative safe and low cost treatments for moderate infectious diarrhea. In this study, Bifidobacterium bifidum G9-1 (BBG9-1), which has been used as an intestinal drug for several decades, was shown to have a remarkable protective effect against RV gastroenteritis in a suckling mice model. As well as prophylactic oral administration of BBG9-1 from 2 days before RV infection, therapeutic oral administration of BBG9-1 from 1 day after RV infection significantly alleviated RV-induced diarrhea. Therapeutic administration of BBG9-1 reduced various types of damage in the small intestine, such as epithelial vacuolization and villous shortening, and significantly diminished the infectious RV titer in mixtures of cecal contents and feces. It was also shown that therapeutic administration of BBG9-1 significantly increased the number of acidic mucin-positive goblet cells and the gene expression of mucosal protective factors including MUC2, MUC3, MUC4, TGFß1 and TFF3 in the small intestine. This led to alleviation of low gut permeability shown as decreased gene expression levels of occludin, claudin-1 and villin-1 after RV infection. Furthermore, in the small intestine, therapeutic administration of BBG9-1 significantly palliated the decreased gene expression of SGLT-1, which plays an important role in water absorption. In the large intestine, administered BBG9-1 was shown to replicate to assimilate undigested nutrients, resulting in normalization of the abnormally high osmotic pressure. These results suggested that water malabsorption caused by RV infection was alleviated in mice administered BBG9-1. Thus, the present study showed that oral administration of BBG9-1 palliated diarrhea partly through protection against RV-induced lesions by inducing mucosal protective factors. Oral administration of BBG9-1 is thought to be an efficient method for management of an RV epidemic for both prophylactic and therapeutic purposes.