Efficacy of Selected Live Biotherapeutic Candidates to Inhibit the Interaction of an Adhesive-Invasive Escherichia coli Strain with Caco-2, HT29-MTX Cells and Their Co-Culture.

Adherent-invasive Escherichia coli (AIEC) has been implicated as a microbiological factor in the pathogenesis of inflammatory bowel disease (IBD). We evaluated the ability of six live biotherapeutic products (LBPs) to inhibit the interaction of an AIEC strain to three cell lines representing human gut epithelium. Co-inoculation of LBPs with AIEC showed a reduction in adhesion (up to 73%) and invasion of AIEC (up to 89%). Pre-inoculation of LBPs in HT-29-MTX and Caco-2 cells before challenging with AIEC further reduced the adhesion and invasion of the AIEC, with three LBPs showing significantly (p < 0.0001) higher efficiency in reducing the adhesion of AIEC. In co-inoculation experiments, the highest reduction in adhesion (73%) of AIEC was observed in HT-29-MTX cells, whereas the highest reduction in invasion (89%) was seen in HT-29-MTX and the co-culture of cells. Pre-inoculation of LBPs further reduced the invasion of AIEC with highest reduction (97%) observed in co-culture of cells. Our results indicated that whilst there were differences in the efficacy of LBPs, they all reduced interaction of AIEC with cell lines representing gut epithelium. Their efficiency was higher when they were pre-inoculated onto the cells, suggesting their potential as candidates for alleviating pathogenesis of AIEC in patients with IBD.

Targeting the Intestinal Microbiota to Prevent Type 2 Diabetes and Enhance the Effect of Metformin on Glycaemia: A Randomised Controlled Pilot Study.

Early treatment may prevent or delay the onset of type 2 diabetes mellitus (T2DM) in individuals who are at high risk. Lifestyle interventions and the hypoglycemic drug metformin have been shown to reduce T2DM incidence. The effectiveness of such interventions may be enhanced by targeting environmental factors such as the intestinal microbiota, which has been proven to predict the response to lifestyle interventions and play a part in mediating the glucose-lowering effects of metformin. Shifts in the intestinal microbiota "towards a more balanced state" may promote glucose homeostasis by regulating short-chain fatty acids' production. This study aimed to investigate the safety and effect of a multi-strain probiotic on glycemic, inflammatory, and permeability markers in adults with prediabetes and early T2DM and to assess whether the probiotic can enhance metformin's effect on glycaemia. A randomised controlled pilot study was conducted in 60 adults with a BMI ≥ 25 kg/m2 and with prediabetes or T2DM (within the previous 12 months). The participants were randomised to a multi-strain probiotic (L. plantarum, L. bulgaricus, L. gasseri, B. breve, B. animalis sbsp. lactis, B. bifidum, S. thermophilus, and S. boulardii) or placebo for 12 weeks. Analyses of the primary outcome (fasting plasma glucose) and secondary outcomes, including, but not limited to, circulating lipopolysaccharide, zonulin, and short chain fatty acids and a metagenomic analysis of the fecal microbiome were performed at baseline and 12 weeks post-intervention. The results showed no significant differences in the primary and secondary outcome measures between the probiotic and placebo group. An analysis of a subgroup of participants taking metformin showed a decrease in fasting plasma glucose, HbA1c, insulin resistance, and zonulin; an increase in plasma butyrate concentrations; and an enrichment of microbial butyrate-producing pathways in the probiotic group but not in the placebo group. Probiotics may act as an adjunctive to metformin by increasing the production of butyrate, which may consequently enhance glucose management.

The effect of a novel probiotic on metabolic biomarkers in adults with prediabetes and recently diagnosed type 2 diabetes mellitus: study protocol for a randomized controlled trial.

BACKGROUND: Shifts in the gastrointestinal microbiome have been shown to contribute to the progression of metabolic diseases including prediabetes and type 2 diabetes mellitus. Research suggests that in-vivo modulation of the gut microbiome by specific probiotic microorganisms may improve insulin sensitivity and blood sugar management, preventing or delaying the development of type 2 diabetes mellitus. However, further research is needed to understand the effect of probiotics as a therapy for the treatment of metabolic diseases. An evidence-based multi-species probiotic was developed to encourage a shift in the gastrointestinal bacterial cohort from a disease-prone to a balanced state with the aim of improving metabolic markers associated with type 2 diabetes mellitus. METHODS: Sixty adults with a body mass index ≥25 kg/m2 with prediabetes or type 2 diabetes mellitus (diagnosed within the previous 12 months) will be enrolled in a double-blind, placebo-controlled pilot study. Participants will be randomized to a multi-species probiotic or placebo for 12 weeks. Both groups will receive lifestyle and nutritional advice. The primary outcome measure is the change between groups in fasting plasma glucose levels from baseline to 12 weeks. Secondary outcome measures include, but are not limited to, the change in lipid profile, systemic inflammation, gut permeability, and faecal microbial and metabolomic profiles. Blood and stool samples are collected at baseline and 12 weeks after treatment. DISCUSSION: Intentional manipulation of gastrointestinal microbial profiles may be useful for preventing and controlling type 2 diabetes mellitus and its associated metabolic complications. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry, ACTRN12613001378718 . Registered on 16 December 2013.

Probiotics, D-Lactic acidosis, oxidative stress and strain specificity.

The existence of an implicit living microscopic world, composed primarily of bacteria, has been known for centuries. The exact mechanisms that govern the contribution of bacteria to human health and disease have only recently become the subject of intense research efforts. Within this very evident shift in paradigms, the rational design of probiotic formulations has led to the creation of an industry that seeks to progress the engineering of probiotic bacteria that produce metabolites that may enhance human host health and prevent disease. The promotion of probiotics is often made in the absence of quality scientific and clinically plausible data. The latest incursions into the probiotic market of claims have posited the amelioration of oxidative stress via potent antioxidant attributes or limiting the administration of probiotics to those species that do not produce D-Lactic acid (i.e., claims that D-Lactic acid acidosis is linked to chronic health conditions) or are strain-specific (shaping an industry point of difference) for appraising a therapeutic effect. Evidence-based research should guide clinical practice, as there is no place in science and medicine that supports unsubstantiated claims. Extravagant industry based notions continue to fuel the imprimatur of distrust and skepticism that is leveled by scientists and clinicians at an industry that is already rife with scientific and medical distrust and questionable views on probiotics. Ignoring scientifically discordant data, when sorting through research innovations and false leads relevant to the actions of probiotics, drives researcher discomfit and keeps the bar low, impeding the progress of knowledge. Biologically plausible posits are obligatory in any research effort; companies formulating probiotics often exhibit a lack of analytical understanding that then fuels questionable investigations failing to build on research capacity.

Gastrointestinal Tract Commensal Bacteria and Probiotics: Influence on End-Organ Physiology.

Bacteria represent the earliest form of independent life on this planet. Bacterial development has included cooperative symbiosis with plants (e.g., Leguminosae family and nitrogen fixing bacteria in soil) and animals (e.g., the gut microbiome). It is generally agreed upon that the fusion of two prokaryotes evolutionarily gave rise to the eukaryotic cell in which mitochondria may be envisaged as a genetically functional mosaic, a relic from one of the prokaryotes. This is expressed by the appearance of mitochondria in eukaryotic cells (an alpha-proteobacteria input), a significant endosymbiotic evolutionary event. As such, the evolution of human life has been complexly connected to bacterial activities. Hence, microbial colonization of mammals has been a progressively driven process. The interactions between the human host and the microbiome inhabiting the gastrointestinal tract (GIT) for example, afford the human host the necessary cues for the development of regulated signals that in part are induced by reactive oxygen species (ROS). This regulated activity then promotes immunological tolerance and metabolic regulation and stability, which then helps establish control of local and extraintestinal end-organ (e.g., kidneys) physiology. Pharmacobiotics, the targeted administration of live probiotic cultures, is an advancing area of potential therapeutics, either directly or as adjuvants. Hence the continued scientific understanding of the human microbiome in health and disease may further lead to fine tuning the targeted delivery of probiotics for a therapeutic gain.

Perna canaliculus (Green-Lipped Mussel): Bioactive Components and Therapeutic Evaluation for Chronic Health Conditions.

Perna canaliculus (Green-Lippped Mussel) is found only in New Zealand waters and is cultivated and manufactured for both the food and nutraceutical industry world-wide. P. canaliculus has traditionally been used as a therapeutic to treat various arthralgias in both humans and animals; however, clinical research reports provide conflicting results. Numerous in vitro studies have reported anti-inflammatory activity of the mussel under various conditions and also demonstrated a synergistic effect with pharmaceutical medications such as non-steroidal anti-inflammatory drugs (NSAIDs) with P. canaliculus protecting the gastrointestinal mucosal lining against such medications. It is proposed that the anti-inflammatory activity demonstrated by P. canaliculus is predominantly due to the lipid fraction, however, among the major classes of compounds found in mussel meat, proteins and peptides are the largest with isolates demonstrating various anti-microbial, anti-inflammatory, anti-oxidant, bioadhesive and anti-hypertensive activities. A review of the bioactive components, their function and therapeutic application is outlined in this chapter. Furthermore, we hypothesise and provide supportive evidence that the gastrointestinal microbiota play an important role in disease processes such as Rheumatoid arthritis and Osteoarthritis and also in the efficacy of P. canaliculus in chronic inflammatory conditions. The metabolic capacity of intestinal microbiota can modify bioactive food components altering the hosts' exposure to these components, potentially enhancing or diminishing their health effects. Understanding the interaction of the bioactive compounds in P. canaliculus with commensal and pathogenic bacteria may facilitate the development of novel interventions to control intestinal and extraintestinal inflammation.

Metabolic Interactions in the Gastrointestinal Tract (GIT): Host, Commensal, Probiotics, and Bacteriophage Influences.

Life on this planet has been intricately associated with bacterial activity at all levels of evolution and bacteria represent the earliest form of autonomous existence. Plants such as those from the Leguminosae family that form root nodules while harboring nitrogen-fixing soil bacteria are a primordial example of symbiotic existence. Similarly, cooperative activities between bacteria and animals can also be observed in multiple domains, including the most inhospitable geographical regions of the planet such as Antarctica and the Lower Geyser Basin of Yellowstone National Park. In humans bacteria are often classified as either beneficial or pathogenic and in this regard we posit that this artificial nomenclature is overly simplistic and as such almost misinterprets the complex activities and inter-relationships that bacteria have with the environment as well as the human host and the plethora of biochemical activities that continue to be identified. We further suggest that in humans there are neither pathogenic nor beneficial bacteria, just bacteria embraced by those that tolerate the host and those that do not. The densest and most complex association exists in the human gastrointestinal tract, followed by the oral cavity, respiratory tract, and skin, where bacteria-pre- and post-birth-instruct the human cell in the fundamental language of molecular biology that normally leads to immunological tolerance over a lifetime. The overall effect of this complex output is the elaboration of a beneficial milieu, an environment that is of equal or greater importance than the bacterium in maintaining homeostasis.

The overarching influence of the gut microbiome on end-organ function: the role of live probiotic cultures.

At the time of birth, humans experience an induced pro-inflammatory beneficial event. The mediators of this encouraged activity, is a fleet of bacteria that assault all mucosal surfaces as well as the skin. Thus initiating effects that eventually provide the infant with immune tissue maturation. These effects occur beneath an emergent immune system surveillance and antigenic tolerance capability radar. Over time, continuous and regulated interactions with environmental as well as commensal microbial, viral, and other antigens lead to an adapted and maintained symbiotic state of tolerance, especially in the gastrointestinal tract (GIT) the organ site of the largest microbial biomass. However, the perplexing and much debated surprise has been that all microbes need not be targeted for destruction. The advent of sophisticated genomic techniques has led to microbiome studies that have begun to clarify the critical and important biochemical activities that commensal bacteria provide to ensure continued GIT homeostasis. Until recently, the GIT and its associated micro-biometabolome was a neglected factor in chronic disease development and end organ function. A systematic underestimation has been to undervalue the contribution of a persistent GIT dysbiotic (a gut barrier associated abnormality) state. Dysbiosis provides a plausible clue as to the origin of systemic metabolic disorders encountered in clinical practice that may explain the epidemic of chronic diseases. Here we further build a hypothesis that posits the role that subtle adverse responses by the GIT microbiome may have in chronic diseases. Environmentally/nutritionally/and gut derived triggers can maintain microbiome perturbations that drive an abnormal overload of dysbiosis. Live probiotic cultures with specific metabolic properties may assist the GIT microbiota and reduce the local metabolic dysfunctions. As such the effect may translate to a useful clinical treatment approach for patients diagnosed with a metabolic disease for end organs such as the kidney and liver. A profile emerges that shows that bacteria are diverse, abundant, and ubiquitous and have significantly influenced the evolution of the eukaryotic cell.

Probiotics, prebiotics and the gastrointestinal tract in health and disease.

The microbiome located in the human gastrointestinal tract (GIT) comprises the largest community (diverse and dense) of bacteria, and in conjunction with a conducive internal milieu, promotes the development of regulated pro- and anti-inflammatory signals within the GIT that promotes immunological and metabolic tolerance. In addition, host-microbial interactions govern GIT inflammation and provide cues for upholding metabolic regulation in both the host and microbes. Failure to regulate inflammatory responses can increase the risk of developing inflammatory conditions in the GIT. Here, we review clinical studies regarding the efficacy of probiotics/prebiotics and the role they may have in restoring host metabolic homeostasis by rescuing the inflammatory response. The clinical studies reviewed included functional constipation, antibiotic-associated diarrhoea, Clostridium difficile diarrhoea, infectious diarrhoea/gastroenteritis, irritable bowel syndrome, inflammatory bowel diseases and necrotizing enterocolitis. We have demonstrated that there was an overall reduction in risk when probiotics were administered over placebo in the majority of GIT inflammatory conditions. The effect size of a cumulative reduction in relative risk for the GIT conditions/diseases investigated was 0.65 (0.61-0.70) (z = 13.3); p < 0.0001 that is an average reduction in risk of 35 % in favour of probiotics. We also progress a hypothesis that the GIT comprises numerous micro-axes (e.g. mucus secretion, Th1/Th2 balance) that are in operational homeostasis; hence probiotics and prebiotics may have a significant pharmacobiotic regulatory role in maintaining host GIT homeostasis in disease states partially through reactive oxygen species signalling.

A phase II randomised double-blind placebo-controlled clinical trial investigating the efficacy and safety of ProstateEZE Max: a herbal medicine preparation for the management of symptoms of benign prostatic hypertrophy.

OBJECTIVE: The aim of the clinical trial was to evaluate the efficacy and safety of ProstateEZE Max, an orally dosed herbal preparation containing Cucurbita pepo, Epilobium parviflorum, lycopene, Pygeum africanum and Serenoa repens in the management of symptoms of medically diagnosed benign prostate hypertrophy (BPH). DESIGN: This was a short-term phase II randomized double-blind placebo controlled clinical trial. SETTING: The trial was conducted on 57 otherwise healthy males aged 40-80 years that presented with medically diagnosed BPH. INTERVENTION: The trial participants were assigned to receive 3 months of treatment (1 capsule per day) with either the herbal preparation (n = 32) or a matched placebo capsule (n = 25). OUTCOME MEASURES: The primary outcome measure was the international prostate specific score (IPSS) measured at baseline, 1, 2 and 3 months. The secondary outcomes were the specific questions of the IPSS and day-time and night-time urinary frequency. RESULTS: There was a significant reduction in IPSS total median score in the active group of 36% as compared to 8% for the placebo group, during the 3-months intervention (p < 0.05). The day-time urinary frequency in the active group also showed a significant reduction over the 3-months intervention (7.0-5.9 times per day, a reduction of 15.6% compared to no significant reduction change for the placebo group (6.2-6.3 times per day) (p < 0.03). The night-time urinary frequency was also significantly reduced in the active group (2.9-1.8, 39.3% compared to placebo (2.8-2.6 times, 7%) (p < 0.004). CONCLUSION: The herbal preparation (ProstateEZE Max) was shown to be well tolerated and have a significant positive effect on physical symptoms of BPH when taken over 3 months, a clinically significant outcome in otherwise healthy men.

The clinical efficacy of a bovine lactoferrin/whey protein Ig-rich fraction (Lf/IgF) for the common cold: a double blind randomized study.

OBJECTIVE: The aim of the study was to determine if a bovine lactoferrin/whey protein Ig-rich fraction (Lf/IgF) combination was effective in reducing the number of colds and in turn improving symptom recovery in a cohort of males and females that reported frequently contracting a cold. DESIGN: A double blind randomized placebo-controlled clinical trial. SETTING: One-hundred and twenty-six participants matched by age, BMI, dietary and physical parameters with self-reported frequent upper respiratory tract symptoms and infections were randomly assigned to receive 600 mg of Lf/IgF or a placebo daily for 90 days. MAIN OUTCOME MEASURES AND RESULTS: A total of 90 participants (47 receiving the active and 43 placebo) completed the 90 day trial and 15 completed 45 days participation (6 in the active and 9 in the placebo group). The total number of colds recorded over the study period was 48 for the treatment group versus 112 for the placebo group (p < 0.001). The significant trend was retained when the data was corrected for medications returned (p < 0.001) and for guessing treatment allocations (p < 0.001). Non-parametric analysis demonstrated that the total number of cold-associated symptoms reported by participants that received Lf/IgF was significantly less than those in the placebo group (p < 0.05). Also, total days sick with a cold and cold severity were reduced over the clinical trial period for Lf/IgF over placebo, but the trend was not significant. CONCLUSIONS: These findings demonstrate that the Lf/IgF combination significantly decreased the incidence of colds and the cumulative number of cold-related symptoms over placebo. This therapeutic combination may be indicated for the prevention of colds and its most common symptoms in the general population when administered as a preventative supplement.

The gastrointestinal microbiome and musculoskeletal diseases: a beneficial role for probiotics and prebiotics.

Natural medicines are an attractive option for patients diagnosed with common and debilitating musculoskeletal diseases such as Osteoarthritis (OA) or Rheumatoid Arthritis (RA). The high rate of self-medication with natural products is due to (1) lack of an available cure and (2) serious adverse events associated with chronic use of pharmaceutical medications in particular non-steroidal anti-inflammatory drugs (NSAIDs) and high dose paracetamol. Pharmaceuticals to treat pain may disrupt gastrointestinal (GIT) barrier integrity inducing GIT inflammation and a state of and hyper-permeability. Probiotics and prebiotics may comprise plausible therapeutic options that can restore GIT barrier functionality and down regulate pro-inflammatory mediators by modulating the activity of, for example, Clostridia species known to induce pro-inflammatory mediators. The effect may comprise the rescue of gut barrier physiological function. A postulated requirement has been the abrogation of free radical formation by numerous natural antioxidant molecules in order to improve musculoskeletal health outcomes, this notion in our view, is in error. The production of reactive oxygen species (ROS) in different anatomical environments including the GIT by the epithelial lining and the commensal microbe cohort is a regulated process, leading to the formation of hydrogen peroxide which is now well recognized as an essential second messenger required for normal cellular homeostasis and physiological function. The GIT commensal profile that tolerates the host does so by regulating pro-inflammatory and anti-inflammatory GIT mucosal actions through the activity of ROS signaling thereby controlling the activity of pathogenic bacterial species.

Green-lipped mussel extract (Perna canaliculus) and glucosamine sulphate in patients with knee osteoarthritis: therapeutic efficacy and effects on gastrointestinal microbiota profiles.

OBJECTIVE: To investigate how changes in the gastrointestinal tract (GIT) microbiota profile may influence nutraceutical efficacy in osteoarthritis (OA) and allow the formulation of a hypothesis that explains in part the inconsistent and contentious findings from OA clinical studies with green-lipped mussel (GLM) and glucosamine. METHODS: A non-blinded randomised clinical trial was conducted with 38 subjects diagnosed with knee OA. Each participant received either 3,000 mg/day of a whole GLM extract or 3,000 mg/day of glucosamine sulphate (GS), p.o. for 12 weeks. Faecal microbial analyses were carried out after collecting stools at T (0) and T (12) weeks. Additional pharmacometric measures were obtained from changes in arthritic scores in the Western Ontario McMaster Universities Arthritis Index (WOMAC) and the Lequesne algofunctional indices and the Gastrointestinal Symptom Rating Scale (GSRS). An intention-to-treat analysis was employed and participant data collected at T (0), T (6) and T (12) weeks. RESULTS: There were no statistically significant changes in bacterial growth patterns determined by the Wilcoxon test. In both groups there was a trend towards a decrease in Clostridium and Staphylococcus species and increase in Lactobacillus, Streptococcus and Eubacterium species. In the GLM group Bifidobacterium tended to increase and Enterococcus and yeast species to decrease. The GS-treated group demonstrated a trend towards a decrease in Bacteroides and an increase in yeasts and Coliforms species, most notably Escherichia coli. We further confirm significant improvement (p < 0.05) in all OA outcome measures from T (0) to T (12) weeks for both the GLM and GS groups. The GSRS scores indicated that GIT function significantly improved over the 12 weeks duration with GLM and GS supplementation. CONCLUSION: Both GLM and GS reduced OA symptoms and non-significantly altered the gut microbiota profile from baseline. Changes in the microbiota profiles occurred in both treatment groups; the most notable being a reduction in the Clostridia sp. This study suggests that nutritional supplements such as GLM and GS may regulate some of the metabolic and immunological activities of the GIT microbiota. The decrease in Clostridia, a potent modulator of colonic Th17 and CD4+ regulatory T cells, was consistent with a decrease in inflammation; improved GSRS scores and OA symptoms for these OA participants. The GIT microbiota may be important factor in the first-pass metabolism of these nutraceuticals.

Green-lipped mussel (Perna canaliculus) extract efficacy in knee osteoarthritis and improvement in gastrointestinal dysfunction: a pilot study.

OBJECTIVE: Clinical data demonstrating efficacy for nutraceutical compounds marketed for the symptom relief of osteoarthritis (OA) have been largely contentious. Furthermore, no association has been linked between clinical trial inconsistencies and gastrointestinal (GI) dysfunction. The aim of this study was to primarily investigate the efficacy of a high-dose New Zealand green-lipped mussel (GLM) extract in patients diagnosed with OA of the knee and concurrently assess GLM impact on GI function. METHODS: An open label, single group allocation study was conducted, that administered 3,000 mg/day of GLM extract over 8 weeks to 21 subjects diagnosed with knee OA. Outcome measures were scored using the WOMAC, the Lequesne algofunctional index, and the Gastrointestinal Symptom Rating Scale (GSRS) tools. An intention-to-treat analysis was employed and subject data collected at T0, T4 and T8 weeks. RESULTS: Paired t tests showed significant improvement for the Lequesne, WOMAC (p < 0.001) and GSRS (p = 0.005) scores. A repeated measures ANOVA analysis showed significant improvement in scores for the Lequesne (F = 20.317, p < 0.001), WOMAC (F = 28.383, p < 0.001) and the GSRS (F = 9.221, p = 0.002). CONCLUSION: Green-lipped mussel significantly improved knee joint pain, stiffness and mobility. We report for the first time that the administration of GLM extract also significantly improved GI symptoms by 49% in OA patients. Given that GI dysfunction is linked to analgesic medication use, we further conclude that the therapeutic efficacy of the GLM extract used was possibly correlated to its effects on GI function by improving GSRS scores from baseline. Results from this trial highlight the requisite for further clinical investigations of gastrointestinal tract function in OA patients.