Effects of Brassica on the human gut microbiota

Brassica vegetables, such as broccoli, are characterised by the presence of sulphur-containing compounds, termed glucosinolates, which are associated with potential health benefits for humans. Glucosinolates are metabolised in the gut by members of the gut microbiota, producing biologically active breakdown products, such as isothiocyanates. The effects of consuming Brassica on the composition of the gut microbiota, and the bacterial mechanisms employed for glucosinolate metabolism, are unclear, and forms the basis of the research presented in this thesis. Culturing human faecal microbiotas in an in vitro batch fermentation model identified the bacterial-mediated reduction of glucoraphanin and glucoiberin to glucoerucin and glucoiberverin, respectively. An Escherichia coli strain was found to exhibit reductase activity on glucoraphanin and the broccoli-derived compound S-methylcysteine sulphoxide, through the reduction of the sulphoxide moiety. Within this fermentation model, the relative proportions of members of the genus Lactobacillus were found to significantly increase when the microbiota was repeatedly exposed to a broccoli leachate, and 16S rDNA sequencing identified these as L. fermentum. Metabolite analysis detected relatively high concentrations of lactate and short-chain fatty acids when faecal microbiotas were cultured in the presence of broccoli leachates, compared to a glucose control media. A human dietary study investigating the effects of Brassica on the microbiota composition revealed a significant association between dietary Brassica and changes to the relative proportions of a number of bacteria, many of which belong to the Clostridiales. Further studies are required to reveal the nature of this association, and whether the presence of glucosinolates may have been a factor. The work presented in this thesis highlights the strong connection between diet, the gut microbiota, and the potential health benefits to the host that may be derived from the bacterial metabolism of dietary compounds

A decrease in iron availability to human gut microbiome reduces the growth of potentially pathogenic gut bacteria: an in vitro colonic fermentation study

Iron-supplements are widely consumed; however most of the iron is not absorbed and enters the colon where potentially pathogenic bacteria can utilise it for growth. This study investigated the effect of iron availability on human gut microbial composition and function using an in vitro colonic fermentation model inoculated with faecal microbiota from healthy adult donors, as well as examining the effect of iron on the growth of individual gut bacteria. Batch fermenters were seeded with fresh faecal material and supplemented with the iron chelator, bathophenanthroline disulphonic acid (BPDS). Samples were analysed at regular intervals to assess impact on the gut bacterial communities. The growth of Escherichia coli and Salmonella Typhimurium was significantly impaired when cultured independently in iron-deficient media. In contrast, depletion of iron did not affect the growth of the beneficial species, Lactobacillus rhamnosus, when cultured independently. Analysis of the microbiome composition via 16S-based metataxonomics indicated that under conditions of iron chelation, the relative abundance decreased for several taxa, including a 10% decrease in Escherichia and a 15% decrease in Bifidobacterium. Metabolomics analysis using 1 HNMR indicated that the production of SCFAs was reduced under iron-limited conditions. These results support previous studies demonstrating the essentiality of iron for microbial growth and metabolism, but, in addition, they indicate that iron chelation changes the gut microbiota profile and influences human gut microbial homeostasis through both compositional and functional changes

The divergent restoration effects of Lactobacillus strains in antibiotic-induced dysbiosis

To evaluate functions of Lactobacillus strains, isolated from fermented food, in restoration of ampicillin-induced disruption based on mucosal barrier, gut microbial community and metabolome analyses, three Lactobacillus strains, L. plantarum CGMCC12436 (LacP), L. casei CGMCC 12,435 (LacC) and L. rhamnosus strain GG (LacG) were individually administered to ampicillin-pretreated mice. All three strains significantly restored concentrations of endotoxin and diamine oxidase to control levels. Linear discriminate analysis based on 16S rRNA sequencing of faecal bacteria revealed that the restoration of microbial communities by Lactobacillus strains was more effective than natural restoration. Correlation analysis between microbiota and metabolites indicated that, the higher level of acetate in LacC group was positively correlated with increased relative abundance of Citrobacter, Bifidobacterium and S24-7. Furthermore, LacC down-regulated the expression of NF-κB p65 and modulated the ampicillin-induced inflammatory responses. The LacC strain could particularly attenuate ampicillin-induced disruption by optimisation of microbial taxa and enhancement of acetate and butyrate production

Comparison of Different Strategies for Providing Fecal Microbiota Transplantation to Treat Patients with Recurrent Clostridium difficile Infection in Two English Hospitals: A Review

Fecal microbiota transplant (FMT) has emerged as a highly efficacious treatment for difficult cases of refractory and/or recurrent Clostridium difficile infection (CDI). There have been many well-conducted randomized controlled trials and thousands of patients reported in case series that describe success rates of approximately 90% following one or more FMT. Although the exact mechanisms of FMT have yet to be fully elucidated, replacement or restoration of a ‘normal’ microbiota (or at least a microbiota resembling those who have never had CDI) appears to have a positive effect on the gut dysbiosis that is thought to exist in these patients. Furthermore, despite being aesthetically unappealing, this ‘ultimate probiotic’ is a particularly attractive solution to a difficult problem that avoids repeated courses of antibiotics. The lack of clarity about the exact mechanism of action and the ‘active ingredient’ of FMT (e.g., individual or communities of bacteria, bacteriophage, or bioactive molecules such as bile acids) has hindered the ability to produce a standardized and well-characterized FMT product. There is no standard method to produce material for FMT, and there are a multitude of factors that can vary between institutions that offer this therapy. Only a few studies have directly compared clinical efficacy in groups of patients who have been treated with FMT prepared differently (e.g., fresh vs. frozen) or administered by different route (e.g., by nasojejunal tube, colonoscopy or by oral administration of encapsulated product). More of these studies should be undertaken to clarify the superiority or otherwise of these variables. This review describes the methods and protocols that two English NHS hospitals independently adopted over the same time period to provide FMT for patients with recurrent CDI. There are several fundamental differences in the methods used, including selection and testing of donors, procedures for preparation and storage of material, and route of administration. These methods are described in detail in this review highlighting differing practice. Despite these significant methodological variations, clinical outcomes in terms of cure rate appear to be remarkably similar for both FMT providers. Although both hospitals have treated only modest numbers of patients, these findings suggest that many of the described differences may not be critical factors in influencing the success of the procedure. As FMT is increasingly being proposed for a number of conditions other than CDI, harmonization of methods and techniques may be more critical to the success of FMT, and thus it will be important to standardize these as far as practically possible

APOE genotype influences the gut microbiome structure and function in humans and mice: relevance for Alzheimer’s disease pathophysiology

Apolipoprotein E (APOE) genotype is the strongest prevalent genetic risk factor for Alzheimer’s disease (AD). Numerous studies have provided insights into the pathologic mechanisms. However, a comprehensive understanding of the impact ofAPOEgenotype onmicroflora speciation and metabolismis completely lacking. In this study,we investigated the association between APOE genotype and the gut microbiome composition in human and APOE–targeted replacement (TR) transgenic mice. Fecal microbiota amplicon sequencing from matched individuals with different APOE genotypes revealed no significant differences in overall microbiota diversity in group aggregated human APOE genotypes. However, several bacterial taxa showed significantly different relative abundance between APOE genotypes. Notably, we detected an association of Prevotellaceae and Ruminococcaceae and several butyrate-producing genera abundances with APOE genotypes. These findings were confirmed by comparing the gutmicrobiota ofAPOE-TRmice. Furthermore, metabolomic analysis of murine fecalwater detected significant differences in microbe-associated amino acids and short-chain fatty acids between APOE genotypes. Together, these findings indicate that APOE genotype is associated with specific gut microbiome profiles in both humans and APOE-TR mice. This suggests that the gut microbiome is worth further investigation as a potential target to mitigate the deleterious impact of the APOE4 allele on cognitive decline and the prevention of A

Refined diet consumption increases neuroinflammatory signalling through bile acid dysmetabolism

Over recent decades, dietary patterns have changed significantly due to the increasing availability of convenient, ultra-processed refined foods. Refined foods are commonly depleted of key bioactive compounds, which have been associated with several deleterious health conditions. As the gut microbiome can influence the brain through a bidirectional communication system known as the ‘microbiota-gut-brain axis’, the consumption of refined foods has the potential to affect cognitive health. In this study, multi-omics approaches were employed to assess the effect of a refined diet on the microbiota-gut-brain axis, with a particular focus on bile acid metabolism. Mice maintained on a refined low-fat diet (rLFD), consisting of high sucrose, processed carbohydrates and low fibre content, for eight weeks displayed significant gut microbial dysbiosis, as indicated by diminished alpha diversity metrics (p < 0.05) and altered beta diversity (p < 0.05) when compared to mice receiving a chow diet. Changes in gut microbiota composition paralleled modulation of the metabolome, including a significant reduction in short-chain fatty acids (acetate, propionate and n-butyrate; p < 0.001) and alterations in bile acid concentrations. Interestingly, the rLFD led to dysregulated bile acid concentrations across both the colon (p < 0.05) and the brain (p < 0.05) which coincided with altered neuroinflammatory gene expression. In particular, the concentration of TCA, TDCA and T-α-MCA was inversely correlated with the expression of NF-κB1, a key transcription factor in neuroinflammation. Overall, our results suggest a novel link between a refined low-fat diet and detrimental neuronal processes, likely in part through modulation of the microbiota-gut-brain axis and bile acid dysmetabolism

Lactobacillus plantarum-mediated regulation of dietary aluminum induces changes in the human gut microbiota: An in vitro colonic fermentation study

The gut microbiota has been identified as a target of toxic metals and a potentially crucial mediator of the bioavailability and toxicity of these metals. In this study, we show that aluminum (Al) exposure, even at low dose, affected the growth of representative strains from the human intestine via pure culture experiments. In vitro, Lactobacillus plantarum CCFM639 could bind Al on its cell surface as shown by electron microscopy and energy dispersive X-ray analysis. The potential of L. plantarum CCFM639 to reverse changes in human intestine microbiota induced by low-dose dietary Al exposure was investigated using an in vitro colonic fermentation model. Batch fermenters were inoculated with fresh stool samples from healthy adult donors and supplemented with 86 mg/L Al and/or 109 CFU of L. plantarum CCFM639. Al exposure significantly increased the relative abundances of Bacteroidetes (Prevotella), Proteobacteria (Escherichia), Actinobacteria (Collinsella), Euryarchaeota (Methanobrevibacter), and Verrucomicrobiaceae and decreased Firmicutes (Streptococcus, Roseburia, Ruminococcus, Dialister, Coprobacillus). Some changes were reversed by the inclusion of L. plantarum CCFM639. Alterations in gut microbiota induced by Al and L. plantarum CCFM639 inevitably led to changes in metabolite levels. The short-chain fatty acid (SCFAs) contents were reduced after Al exposure, but L. plantarum CCFM639 could elevate their levels. SCFAs had positive correlations with beneficial bacteria, such as Dialister, Streptococcus, Roseburia, and negative correlations with Erwinia, Escherichia, and Serratia. Therefore, dietary Al exposure altered the composition and structure of the human gut microbiota, and this was partially mitigated by L. plantarum CCFM639. This probiotic supplementation is potentially a promising and safe approach to alleviate the harmful effects of dietary Al exposure

Systemic iron reduction by venesection alters the gut microbiome in patients with haemochromatosis

Background & Aims: Iron reduction by venesection has been the cornerstone of treatment for haemochromatosis for decades, and its reported health benefits are many. Repeated phlebotomy can lead to a compensatory increase in intestinal iron absorption, reducing intestinal iron availability. Given that most gut bacteria are highly dependent on iron for survival, we postulated that, by reducing gut iron levels, venesection could alter the gut microbiota. Methods: Clinical parameters, faecal bacterial composition and metabolomes were assessed before and during treatment in a group of patients with haemochromatosis undergoing iron reduction therapy. Results: Systemic iron reduction was associated with an alteration of the gut microbiome, with changes evident in those who experienced reduced faecal iron availability with venesection. For example, levels of Faecalibacterium prausnitzii, a bacterium associated with improved colonic health, were increased in response to faecal iron reduction. Similarly, metabolomic changes were seen in association with reduced faecal iron levels. Conclusion: These findings highlight a significant shift in the gut microbiome of patients who experience reduced colonic iron during venesection. Targeted depletion of faecal iron could represent a novel therapy for metabolic and inflammatory diseases, meriting further investigation. Lay summary: Iron depletion by repeated venesection is the mainstay of treatment for haemochromatosis, an iron-overload disorder. Venesection has been associated with several health benefits, including improvements in liver function tests, reversal of liver scarring, and reduced risk of liver cancer. During iron depletion, iron absorption from the gastrointestinal (GI) tract increases to compensate for iron lost with treatment. Iron availability is limited in the GI tract and is crucial to the growth and function of many gut bacteria. In this study we show that reduced iron availability in the colon following venesection treatment leads to a change in the composition of the gut bacteria, a finding that, to date, has not been studied in patients with haemochromatosis

A natural mutation in Pisum sativum L. (pea) alters starch assembly and improves glucose homeostasis in humans

Elevated postprandial glucose (PPG) is a significant risk factor for non-communicable diseases globally. Currently, there is a limited understanding of how starch structures within a carbohydrate-rich food matrix interact with the gut luminal environment to control PPG. Here, we use pea seeds (Pisum sativum) and pea flour, derived from two near-identical pea genotypes (BC1/19RR and BC1/19rr) differing primarily in the type of starch accumulated, to explore the contribution of starch structure, food matrix and intestinal environment to PPG. Using stable isotope 13C-labelled pea seeds, coupled with synchronous gastric, duodenal and plasma sampling in vivo, we demonstrate that maintenance of cell structure and changes in starch morphology are closely related to lower glucose availability in the small intestine, resulting in acutely lower PPG and promotion of changes in the gut bacterial composition associated with long-term metabolic health improvements