Elena M. Comelli

Intestinal microbiota in patients with nonalcoholic fatty liver disease

Despite evidence that the intestinal microbiota (IM) is involved in the pathogenesis of obesity, the IM composition of patients with nonalcoholic fatty liver disease (NAFLD) has not been well characterized. This prospective, cross‐sectional study was aimed at identifying differences in IM between adults with biopsy‐proven NAFLD (simple steatosis [SS] or nonalcoholic steatohepatitis [NASH]) and living liver donors as healthy controls (HC). Fifty subjects were included: 11 SS, 22 NASH, and 17 HC. One stool sample was collected from each participant. Quantitative real‐time polymerase chain reaction was used to measure total bacterial counts, Bacteroides/Prevotella (herein referred to as Bacteroidetes), Clostridium leptum, C. coccoides, bifidobacteria, Escherichia coli and Archaea in stool. Clinical and laboratory data, food records, and activity logs were collected. Patients with NASH had a lower percentage of Bacteroidetes (Bacteroidetes to total bacteria counts) compared to both SS and HC (P = 0.006) and higher fecal C. coccoides compared to those with SS (P = 0.04). There were no differences in the remaining microorganisms. As body mass index (BMI) and dietary fat intake differed between the groups (P < 0.05), we performed linear regression adjusting for these variables. The difference in C. coccoides was no longer significant after adjusting for BMI and fat intake. However, there continued to be a significant association between the presence of NASH and lower percentage Bacteroidetes even after adjusting for these variables (P = 0.002; 95% confidence interval = −0.06 to −0.02).

Adiposity, gut microbiota and faecal short chain fatty acids are linked in adult humans

Background/Objectives:High dietary fibre intakes may protect against obesity by influencing colonic fermentation and the colonic microbiota. Though, recent studies suggest that increased colonic fermentation contributes to adiposity. Diet influences the composition of the gut microbiota. Previous research has not evaluated dietary intakes, body mass index (BMI), faecal microbiota and short chain fatty acid (SCFA) in the same cohort. Our objectives were to compare dietary intakes, faecal SCFA concentrations and gut microbial profiles in healthy lean (LN, BMI⩽25) and overweight or obese (OWOB, BMI>25) participants.Design:We collected demographic information, 3-day diet records, physical activity questionnaires and breath and faecal samples from 94 participants of whom 52 were LN and 42 OWOB.Results:Dietary intakes and physical activity levels did not differ significantly between groups. OWOB participants had higher faecal acetate (P=0.05), propionate (P=0.03), butyrate (P=0.05), valerate (P=0.03) and total short chain fatty acid (SCFA; P=0.02) concentrations than LN. No significant differences in Firmicutes to Bacteroides/Prevotella (F:B) ratio was observed between groups. However, in the entire cohort, Bacteroides/Prevotella counts were negatively correlated with faecal total SCFA (r=−0.32, P=0.002) and F:B ratio was positively correlated with faecal total SCFA (r=0.42, P<0.0001). Principal component analysis identified distinct gut microbiota and SCFA–F:B ratio components, which together accounted for 59% of the variation. F:B ratio loaded with the SCFA and not with the microbiota suggesting that SCFA and F:B ratio vary together and may be interrelated.Conclusions:The results support the hypothesis that colonic fermentation patterns may be altered, leading to different faecal SCFA concentrations in OWOB compared with LN humans. More in-depth studies looking at the metabolic fate of SCFA produced in LN and OWOB participants are needed in order to determine the role of SCFA in obesity.

Altered hepatic gene expression in nonalcoholic fatty liver disease is associated with lower hepatic n‐3 and n‐6 polyunsaturated fatty acids

In nonalcoholic fatty liver disease, hepatic gene expression and fatty acid (FA) composition have been reported independently, but a comprehensive gene expression profiling in relation to FA composition is lacking. The aim was to assess this relationship. In a cross‐sectional study, hepatic gene expression (Illumina Microarray) was first compared among 20 patients with simple steatosis (SS), 19 with nonalcoholic steatohepatitis (NASH), and 24 healthy controls. The FA composition in hepatic total lipids was compared between SS and NASH, and associations between gene expression and FAs were examined. Gene expression differed mainly between healthy controls and patients (SS and NASH), including genes related to unsaturated FA metabolism. Twenty‐two genes were differentially expressed between NASH and SS; most of them correlated with disease severity and related more to cancer progression than to lipid metabolism. Biologically active long‐chain polyunsaturated FAs (PUFAs; eicosapentaenoic acid + docosahexaenoic acid, arachidonic acid) in hepatic total lipids were lower in NASH than in SS. This may be related to overexpression of FADS1, FADS2, and PNPLA3. The degree and direction of correlations between PUFAs and gene expression were different among SS and NASH, which may suggest that low PUFA content in NASH modulates gene expression in a different way compared with SS or, alternatively, that gene expression influences PUFA content differently depending on disease severity (SS versus NASH). Conclusion: Well‐defined subjects with either healthy liver, SS, or NASH showed distinct hepatic gene expression profiles including genes involved in unsaturated FA metabolism. In patients with NASH, hepatic PUFAs were lower and associations with gene expression were different compared to SS. (Hepatology 2015;61:1565–1578)

Digestive and physiologic effects of a wheat bran extract, arabino-xylan-oligosaccharide, in breakfast cereal.

OBJECTIVE We assessed whether a wheat bran extract containing arabino-xylan-oligosaccharide (AXOS) elicited a prebiotic effect and influenced other physiologic parameters when consumed in ready-to-eat cereal at two dose levels. METHODS This double-blind, randomized, controlled, crossover trial evaluated the effects of consuming AXOS at 0 (control), 2.2, or 4.8 g/d as part of ready-to-eat cereal for 3 wk in 55 healthy men and women. Fecal microbial levels, postprandial serum ferulic acid concentrations, and other physiologic parameters were assessed at the beginning and end of each condition. RESULTS The median bifidobacteria content of stool samples (log₁₀/grams of dry weight [DW]) was found to be higher in the subjects consuming the 4.8-g/d dose (10.03) than in those consuming 2.2 g/d (9.93) and control (9.84, P < 0.001). No significant changes in the populations of other fecal microbes were observed, indicating a selective increase in fecal bifidobacteria. Postprandial ferulic acid was measured at 120 min at the start and end of each 3-wk treatment period in subjects at least 50 y old (n = 37) and increased in a dose-dependent manner (end-of-treatment values 0.007, 0.050, and 0.069 μg/mL for the control, AXOS 2.2 g/d, and AXOS 4.8 g/d conditions, respectively, P for trend < 0.001). CONCLUSION These results indicate that AXOS has prebiotic properties, selectively increasing fecal bifidobacteria, and increases postprandial ferulic acid concentrations in a dose-dependent manner in healthy men and women.

Study of the adhesion of Bifidobacterium bifidum MIMBb75 to human intestinal cell lines.

The aim of this study was to investigate the adhesive phenotype of the human intestinal isolate Bifidobacterium bifidum MIMBb75 to human colon carcinoma cell lines. We have previously shown that the adhesion of this strain to Caco-2 cells is mediated by an abundant surface lipoprotein named BopA. In this study, we found that this strain adheres to Caco-2 and HT-29 cells, and that its adhesion strongly depends on the environmental conditions, including the presence of sugars and bile salts and the pH. Considerably more adhesion to a Caco-2 monolayer occurred in the presence of fucose and mannose and less when MIMBb75 grew in Oxgall bile salts compared to standard environmental conditions. In particular, growth in Oxgall bile salts reduced the adhesion ability of MIMBb75 and modified the SDS-PAGE profile of the cell wall associated proteins of the strain. The pH markedly affected both adhesion to Caco-2 and bacterial autoaggregation. Finally, experiments with sodium metaperiodate suggested that not only proteinaceous determinants are involved in the adhesion process of B. bifidum. In conclusion, it seems that the colonization strategy of this bacterium can be influenced by factors varying along the gastrointestinal tract, such as the presence of specific sugars and bile salts and the pH, possibly limiting the adhesion of B.bifidum to only restricted distal sites of the gut.

Bile Acids and Dysbiosis in Non-Alcoholic Fatty Liver Disease.

Background & Aims Non-alcoholic fatty liver disease (NAFLD) is characterized by dysbiosis. The bidirectional effects between intestinal microbiota (IM) and bile acids (BA) suggest that dysbiosis may be accompanied by an altered bile acid (BA) homeostasis, which in turn can contribute to the metabolic dysregulation seen in NAFLD. This study sought to examine BA homeostasis in patients with NAFLD and to relate that with IM data. Methods This was a prospective, cross-sectional study of adults with biopsy-confirmed NAFLD (non-alcoholic fatty liver: NAFL or non-alcoholic steatohepatitis: NASH) and healthy controls (HC). Clinical and laboratory data, stool samples and 7-day food records were collected. Fecal BA profiles, serum markers of BA synthesis 7-alpha-hydroxy-4-cholesten-3-one (C4) and intestinal BA signalling, as well as IM composition were assessed. Results 53 subjects were included: 25 HC, 12 NAFL and 16 NASH. Levels of total fecal BA, cholic acid (CA), chenodeoxycholic acid (CDCA) and BA synthesis were higher in patients with NASH compared to HC (p<0.05 for all comparisons). The primary to secondary BA ratio was higher in NASH compared to HC (p = 0.004), but ratio of conjugated to unconjugated BAs was not different between the groups. Bacteroidetes and Clostridium leptum counts were decreased in in a subset of 16 patients with NASH compared to 25 HC, after adjusting for body mass index and weight-adjusted calorie intake (p = 0.028 and p = 0.030, respectively). C. leptum was positively correlated with fecal unconjugated lithocholic acid (LCA) (r = 0.526, p = 0.003) and inversely with unconjugated CA (r = -0.669, p<0.0001) and unconjugated CDCA (r = - 0.630, p<0.0001). FGF19 levels were not different between the groups (p = 0.114). Conclusions In adults with NAFLD, dysbiosis is associated with altered BA homeostasis, which renders them at increased risk of hepatic injury.

Impact of Bifidobacterium bifidum MIMBb75 on mouse intestinal microorganisms

Bifidobacterium bifidum MIMBb75 is a recently identified probiotic. However, its distribution along the intestine and impact on resident microbiota is unknown. Herein, we established a quantitative real-time polymerase chain reaction assay targeting the B. bifidum-specific BopA region for the quantification of B. bifidum in feces and used this assay to investigate transit of B. bifidum MIMBb75 through the murine intestine. We also analyzed the consequential impact on resident microbial cohorts. C57BL/6J mice were daily gavaged with 0.2 mL of either sterile PBS or PBS containing 10(8) colony-forming units of B. bifidum MIMBb75 for 2 weeks, after which intestinal contents and fecal samples were analyzed for microbial compositional changes. Bifidobacterium bifidum MIMBb75 was able to transiently colonize the murine intestine, with the predominant niche being the ceco-proximal colonic region. Region-specific effects on host microbiota were observed including decreased levels of Clostridium coccoides in the cecum, increased levels of bifidobacteria in the proximal and distal colon, total bacteria and Clostridium leptum in the proximal colon, and of C. coccoides in the feces. These findings suggest that probiotic properties of B. bifidum MIMBb75 may partially depend on its ability to at least transiently colonize the intestine and impact on the resident microbial communities at various intestinal loci.

Practical approaches to probiotics use

Probiotics are microorganisms exerting beneficial effects on the host. They can be ingested through foods or supplements and their inclusion in these products is regulated in Canada by the Health Canada Health Products and Food Branch. The aim of this article is to summarize current evidence from randomized controlled trials and guidelines from Health Canada, the World Health Organization, and internationally recognized expert committees in the hope that it will help practitioners and professionals recommending probiotics to healthy and diseased patients, with a focus on the Canadian setting. From a general perspective, probiotics can be recommended for prevention of diseases that are associated to altered intestinal ecology. Specifically, they can be recommended for prevention of upper respiratory tract infections and pouchitis, for prevention and management of necrotizing enterocolitis, bacterial vaginosis and antibiotic associated diarrhea, including Clostridium difficile infection, and for treatment of atopic eczema in cows milk allergy and of infectious diarrhea. Additional substantiated probiotic benefits include prevention of hypercholesterolemia, management of constipation, reduction of recurrent urinary tract infections, improvement of irritable bowel syndrome symptoms, and reduction of antibiotics side effects in Helicobacter pylori eradication. Because probiotics are generally recognized as safe and can be removed with antimicrobial agents, their use should be considered in patients of all ages.

Gut Microbiota-bone Axis.

ABSTRACT The gut microbiota (GM) is an important regulator of body homeostasis, including intestinal and extra-intestinal effects. This review focuses on the GM-bone axis, which we define as the effect of the gut-associated microbial community or the molecules they synthesize, on bone health. While research in this field is limited, findings from preclinical studies support that gut microbes positively impact bone mineral density and strength parameters. Moreover, administration of beneficial bacteria (probiotics) in preclinical models has demonstrated higher bone mineralization and greater bone strength. The preferential bacterial genus that has shown these beneficial effects in bone is Lactobacillus and thus lactobacilli are among the best candidates for future clinical intervention trials. However, their effectiveness is dependent on stage of development, as early life constitutes an important time for impacting bone health, perhaps via modulation of the GM. In addition, sex-specific difference also impacts the efficacy of the probiotics. Although auspicious, many questions regarding the GM-bone axis require consideration of potential mechanisms; sex-specific efficacy; effective dose of probiotics; and timing and duration of treatment.

Maternal vitamin D beneficially programs metabolic, gut and bone health of mouse male offspring in an obesogenic environment

Background/Objectives:Vitamin D is an anti-inflammatory nutrient and a determinant of bone health. Some prospective studies suggest that maternal vitamin D status is positively associated with offspring bone mass. We found that serum concentrations of lipopolysaccharide (LPS), an inflammatory molecule related to adiposity, insulin resistance and bone resorption, is lower in healthy mouse offspring exposed to high dietary vitamin D during pregnancy and lactation. LPS reaches the circulation via the gut. This study investigated whether maternal vitamin D programs metabolic, gut and bone health of male offspring in an obesogenic environment.Methods:C57BL/6J dams received an AIN-93G diet with high (H) or low (L) vitamin D during pregnancy and lactation. At weaning, offspring remained on their dam’s vitamin D level (LL or HH) or were switched (LH or HL) and fed a high fat (44.2%) and sucrose (19.8%) diet. Glucose response, adiposity, systemic inflammation (LPS, cytokines), intestinal permeability and mass, strength and microarchitecture of trabecular and cortical bone were assessed in 7-month-old male offsprings.Results:Higher maternal dietary vitamin D resulted in lower intestinal permeability (fecal albumin, P=0.010) and benefited trabecular but not cortical bone structure at the distal femur (higher trabecular number, P=0.022; less trabecular separation, P=0.015) and lumbar vertebra 2 (bone volume/total volume%, P=0.049). Higher maternal and offspring vitamin D resulted in lower fasting glucose (HH versus LL, P=0.039) and serum LPS concentrations (dam diet, P=0.011; pup diet, P=0.002). Higher offspring vitamin D resulted in lower epididymal fat pad relative weight (P=0.006). The serum concentrations of IL-6 and TNF-α did not differ among groups.Conclusions:Maternal dietary vitamin D beneficially programs intestinal permeability and systemic LPS concentration, which is accompanied by stronger trabecular bone in an obesogenic environment. Thus, the gut may mediate vitamin D effects. Moreover, optimizing vitamin D in early life may be critical for later health.