Use of colony-based bacterial strain typing for tracking the fate of Lactobacillus strains during human consumption

<p>Abstract</p> <p>Background</p> <p>The Lactic Acid Bacteria (LAB) are important components of the healthy gut flora and have been used extensively as probiotics. Understanding the cultivable diversity of LAB before and after probiotic administration, and being able to track the fate of administered probiotic isolates during feeding are important parameters to consider in the design of clinical trials to assess probiotic efficacy. Several methods may be used to identify bacteria at the strain level, however, PCR-based methods such as Random Amplified Polymorphic DNA (RAPD) are particularly suited to rapid analysis. We examined the cultivable diversity of LAB in the human gut before and after feeding with two <it>Lactobacillus </it>strains, and also tracked the fate of these two administered strains using a RAPD technique.</p> <p>Results</p> <p>A RAPD typing scheme was developed to genetically type LAB isolates from a wide range of species, and optimised for direct application to bacterial colony growth. A high-throughput strategy for fingerprinting the cultivable diversity of human faeces was developed and used to determine: (i) the initial cultivable LAB strain diversity in the human gut, and (ii) the fate of two <it>Lactobacillus </it>strains (<it>Lactobacillus salivarius </it>NCIMB 30211 and <it>Lactobacillus acidophilus </it>NCIMB 30156) contained within a capsule that was administered in a small-scale human feeding study. The <it>L. salivarius </it>strain was not cultivated from the faeces of any of the 12 volunteers prior to capsule administration, but appeared post-feeding in four. Strains matching the <it>L. acidophilus </it>NCIMB 30156 feeding strain were found in the faeces of three volunteers prior to consumption; after taking the <it>Lactobacillus </it>capsule, 10 of the 12 volunteers were culture positive for this strain. The appearance of both <it>Lactobacillus </it>strains during capsule consumption was statistically significant (p < 0.05).</p> <p>Conclusion</p> <p>We have shown that genetic strain typing of the cultivable human gut microbiota can be evaluated using a high throughput RAPD technique based on single bacterial colonies. Validation of this strategy paves the way for future systematic studies on the fate and efficacy of bacterial probiotics during human clinical trials.</p

Glucosamine hydrochloride but not chondroitin sulfate prevents cartilage degradation and inflammation induced by interleukin-1α in bovine cartilage explants

Objective Glucosamine hydrochloride (GH) and chondroitin sulfate (CS) are commonly used for the treatment of osteoarthritis (OA). The aim of this study was to assess their effects, alone and in combination, on preventing aggrecan degradation and inflammation in an in vitro model of OA. Design To test the effects of GH and/or CS as a preventative treatment, cartilage explants were pretreated with the compound(s) using concentrations that showed no detrimental effect on chondrocyte viability. Interleukin-1α (IL-1α) was added to induce cartilage degradation, supernatant and explants were analyzed for proteoglycan degradation products, aggrecanase mRNA expression and activity, and for the release of inflammatory markers. Results Following treatment with IL-1α, 2 mg/mL dose of GH pretreatment was associated with a reduction of glycosaminoglycan release, reduced generation of the pathological interglobular domain aggrecan catabolites, decreased mRNA levels of ADAMTS-4 and -5 and reduced activity of ADAMTS-4. In contrast, CS alone did not have a significant effect on IL-1α-induced cartilage degradation and the addition of 0.4 mg/mL CS to 2 mg/mL GH did not further inhibit IL-1α-induced activity. Pretreatment with 2 mg/mL GH also reduced the release of inflammatory markers, prostaglandin E2 and nitric oxide induced by IL-1α while CS did not have a significant effect. Conclusions The results suggest that GH prevents cartilage degradation mediated by aggrecanases ADAMTS-4 and -5, and may also reduce inflammation. This could be part of the mechanisms by which GH is effective in maintaining joint integrity and function, and preventing or delaying early symptoms of OA

Lactobacillus plantarum CUL66 can impact cholesterol homeostasis in Caco-2 enterocytes

Hypercholesterolemia drives the development of cardiovascular disease, the leading cause of mortality in western society. Supplementation with probiotics that interfere with cholesterol metabolism may provide a contribution to disease prevention. Lactobacillus plantarum CUL66 (NCIMB 30280) has been assessed in vitro for its ability to impact cholesterol absorption. L. plantarum CUL66 tested positive for bile salt hydrolase activity and the ability to assimilate cholesterol from culture media. RT-qPCR analysis showed that the bacterium significantly decreased the expression of Niemann-Pick C1-like 1 and ATP-binding cassette transporter-1 in polarised Caco-2 cells after 6 h exposure. Conversely, the expression of ATP-binding cassette sub-family G member (ABCG)-5 and ABCG-8, and 3-hydroxy-3-methylglutaryl-CoA reductase were significantly increased. Using a radiolabelled assay, we also observed significant reductions in the uptake and basolateral efflux of cholesterol by Caco-2 cells exposed to L. plantarum CUL66. This in vitro study identified L. plantarum CUL66 as a cholesterol lowering bacteria by highlighting its ability to beneficially regulate multiple in vitro events associated with intestinal cholesterol metabolism and provides evidence of efficacy for its inclusion in future in vivo studies

A unique combination of nutritionally active ingredients can prevent several key processes associated with atherosclerosis in vitro

Atherosclerosis is the underlying cause of cardiovascular disease that leads to more global mortalities each year than any other ailment. Consumption of active food ingredients such as phytosterols, omega-3 polyunsaturated fatty acids and flavanols are known to impart beneficial effects on cardiovascular disease although the combined actions of such agents in atherosclerosis is poorly understood. The aim of this study was to screen a nutritional supplement containing each of these active components for its anti-atherosclerotic effect on macrophages in vitro.The supplement attenuated the expression of intercellular adhesion molecule-1 and macrophage chemoattractant protein-1 in human and murine macrophages at physiologically relevant doses. The migratory capacity of human monocytes was also hindered, possibly mediated by eicosapentaenoic acid and catechin, while the ability of foam cells to efflux cholesterol was improved. The polarisation of murine macrophages towards a pro-inflammatory phenotype was also attenuated by the supplement.The formulation was able to hinder multiple key steps of atherosclerosis development in vitro by inhibiting monocyte recruitment, foam cell formation and macrophage polarisation towards an inflammatory phenotype. This is the first time a combination these ingredients has been shown to elicit such effects and supports its further study in preclinical in vivo models

The kynurenine and serotonin pathway, neopterin and biopterin in depressed children and adolescents: an impact of omega-3 fatty acids, and association with markers related to depressive disorder. A randomized, blinded, prospective study

Depressive disorder is a severe mental condition. In addition to genetic factors, immunological-inflammatory factors, oxidative stress, and disturbances in neurotransmitter metabolism, kynurenine and serotonin pathways may play a role. The exact mechanisms, especially in depressed children and adolescents, are not fully understood. Our primary hypothesis was whether the metabolites of tryptophan degradation in children and adolescents with depressive disorder might be influenced by omega-3 FAs compared to omega-6 FAs during a 12-week supplementation. A secondary hypothesis was to investigate whether tryptophan metabolites in children and adolescents are associated with markers of inflammatory response, oxidative stress, cortisol, and the serum omega-6/omega-3 FA ratio. Metabolites of tryptophan degradation and pteridines, neopterin, and biopterin in urine were analyzed with an HPLC system. Surprisingly, omega-3 FAs stimulated both kynurenine (kynurenine/tryptophan ratio) and serotonin (5-hydroxytryptophan) pathways, whereas omega-6 FAs only increased the kynurenine/tryptophan ratio. Neopterin and biopterin were not different from the healthy controls. Biopterin increased after omega-3 FA supplementation. Serotonin was positively correlated with lipoperoxidation and a marker of oxidative protein damage. Of the monitored tryptophan metabolites, only 5-hydroxyindolacetic acid was positively correlated with the severity of depression, total cholesterol, and negatively with brain-derived neurotrophic factor and glutathione peroxidase. In conclusion, in children and adolescents, both supplemented FAs stimulated the kynurenine pathway (kynurenine/tryptophan ratio) and kynurenine formation. However, the serotonin pathway (5-hydroxytryptophan) was stimulated only by omega-3 FA. Tryptophan metabolism is associated with oxidative stress, inflammation, total cholesterol, and cortisol. We are the first to point out the association between the kynurenine pathway (KYN/TRP ratio) and the omega-6/omega-3 FA ratio. The metabolite 5-HIAA could play a role in the pathophysiology of depressive disorder in children and adolescents.Clinical Trial Registrationhttps://www.isrctn.com/ISRCTN81655012, identifier ISRCTN81655012

Vitamin D associates with improved quality of life in participants with irritable bowel syndrome: outcomes from a pilot trial

Background: Vitamin D deficiency has been associated or implicated with the pathophysiology of the gastrointestinal conditions inflammatory bowel disease and colorectal cancer, as well as with depression. No trials or epidemiology studies to date have investigated a link with irritable bowel syndrome (IBS). A single case report has suggested a benefit in IBS of vitamin D supplementation. We hypothesised that IBS participants with vitamin D insufficiency would benefit from repletion in terms of their IBS symptoms. We undertook a pilot trial to provide data to support a power calculation and to justify a full trial. Methods: This was a randomised, double blinded, three-arm parallel design trial of vitamin D, placebo or a combination of vitamin D and probiotics. Participants were further stratified according to whether they were vitamin D replete or insufficient. Vitamin D status was determined by blood test at baseline and exit; IBS symptoms were assessed by validated questionnaire; dietary intakes were assessed by food frequency questionnaire. Results: A significant proportion of the IBS population were vitamin D deficient, such that the replete stratum could not be adequately recruited. There was a significant association in the baseline data between circulating vitamin D level and quality of life (“How much has IBS affected your life?”). Supplementation significantly improved vitamin D level versus placebo. IBS symptoms were not significantly improved in this pilot, although a power calculation was enabled from the intervention data. Conclusions: The IBS population exhibits significant levels of vitamin D insufficiency and would benefit from screening and possible supplementation. The impact of IBS on quality of life may be reduced by vitamin D level. Future trials should have a sample size of over 9

The multifactorial interplay of diet, the microbiome and appetite control: current knowledge and future challenges

The recent availability of high-throughput nucleic acid sequencing technologies has rapidly advanced approaches to analysing the role of the gut microbiome in governance of human health, including gut health, and also metabolic, cardiovascular and mental health, inter alia. Recent scientific studies suggest that energy intake (EI) perturbations at the population level cannot account for the current obesity epidemic, and significant work is investigating the potential role of the microbiome, and in particular its metabolic products, notably SCFA, predominantly acetate, propionate and butyrate, the last of which is an energy source for the epithelium of the large intestine. The energy yield from dietary residues may be a significant factor influencing energy balance. This review posits that the contribution towards EI is governed by EI diet composition (not just fibre), the composition of the microbiome and by the levels of physical activity. Furthermore, we hypothesise that these factors do not exist in a steady state, but rather are dynamic, with both short- and medium-term effects on appetite regulation. We suggest that the existing modelling strategies for bacterial dynamics, specifically for growth in chemostat culture, are of utility in understanding the dynamic interplay of diet, activity and microbiomic organisation. Such approaches may be informative in optimising the application of dietary and microbial therapy to promote health

A randomised controlled study shows supplementation of overweight and obese adults with lactobacilli and bifidobacteria reduces bodyweight and improves well-being

In an exploratory, block-randomised, parallel, double-blind, single-centre, placebo-controlled superiority study (ISRCTN12562026, funded by Cultech Ltd), 220 Bulgarian participants (30 to 65 years old) with BMI 25–34.9 kg/m2 received Lab4P probiotic (50 billion/day) or a matched placebo for 6 months. Participants maintained their normal diet and lifestyle. Primary outcomes were changes in body weight, BMI, waist circumference (WC), waist-to-height ratio (WtHR), blood pressure and plasma lipids. Secondary outcomes were changes in plasma C-reactive protein (CRP), the diversity of the faecal microbiota, quality of life (QoL) assessments and the incidence of upper respiratory tract infection (URTI). Significant between group decreases in body weight (1.3 kg, p < 0.0001), BMI (0.045 kg/m2, p < 0.0001), WC (0.94 cm, p < 0.0001) and WtHR (0.006, p < 0.0001) were in favour of the probiotic. Stratification identified greater body weight reductions in overweight subjects (1.88%, p < 0.0001) and in females (1.62%, p = 0.0005). Greatest weight losses were among probiotic hypercholesterolaemic participants (−2.5%, p < 0.0001) alongside a significant between group reduction in small dense LDL-cholesterol (0.2 mmol/L, p = 0.0241). Improvements in QoL and the incidence rate ratio of URTI (0.60, p < 0.0001) were recorded for the probiotic group. No adverse events were recorded. Six months supplementation with Lab4P probiotic resulted in significant weight reduction and improved small dense low-density lipoprotein-cholesterol (sdLDL-C) profiles, QoL and URTI incidence outcomes in overweight/obese individuals

Lipid-coated ruthenium dendrimer conjugated with doxorubicin in anti-cancer drug delivery: Introducing protocols

One of the major limitations for the treatment of many diseases is an inability of drugs to cross the cell membrane barrier. Different kinds of carriers are being investigated to improve drug bioavailability. Among them, lipid or polymer-based systems are of special interest due to their biocompatibility. In our study, we combined dendritic and liposomal carriers and analysed the biochemical and biophysical properties of these formulations. Two preparation methods of Liposomal Locked-in Dendrimers (LLDs) systems have been established and compared. Carbosilane ruthenium metallodendrimer was complexed with an anti-cancer drug (doxorubicin) and locked in a liposomal structure, using both techniques. The LLDs systems formed by hydrophilic locking had more efficient transfection profiles and interacted with the erythrocyte membrane better than systems using the hydrophobic method. The results indicate these systems have improved transfection properties when compared to noncomplexed components. The coating of dendrimers with lipids significantly reduced their hemotoxicity and cytotoxicity. The nanometric size, low polydispersity index and reduced positive zeta potential of such complexes made them attractive for future application in drug delivery. The formulations prepared by the hydrophobic locking protocol were not effective and will not be considered furthermore as prospective drug delivery systems. In contrast, the formulations formed by the hydrophilic loading method have shown promising results where the cytotoxicity of LLD systems with doxorubicin was more effective against cancer than normal cells

Probiotics with vitamin C for the prevention of upper respiratory tract symptoms in children aged 3-10 years: randomised controlled trial

In a double-blind, randomised, parallel-group, placebo-controlled study, healthy school children aged 3-10 years received a probiotic based supplement daily for 6 months to assess the impact on the incidence and duration of upper respiratory tract infection (URTI) symptoms. The intervention comprised Lab4 probiotic (Lactobacillus acidophilus CUL21 and CUL60, Bifidobacterium bifidum CUL20 and Bifidobacterium animalis subsp. lactis CUL34) at 12.5 billion cfu/day plus 50 mg vitamin C or a matching placebo. 171 children were included in the analysis (85 in placebo and 86 in active group). Incidence of coughing was 16% (P=0.0300) significantly lower in the children receiving the active intervention compared to the placebo. No significant differences in the incidence rate of other URTI symptoms were observed. There was significantly lower risk of experiencing five different URTI related symptoms in one day favouring the active group (Risk ratio: 0.31, 95% confidence interval: 0.12, 0.81, P=0.0163). Absenteeism from school and the use of antibiotics was also significantly reduced for those in the active group (-16%, P=0.0060 and -27%, P=0.0203, respectively). Our findings indicate that six months daily supplementation with the Lab4 probiotic and vitamin C combination reduces the incidence of coughing, absenteeism and antibiotic usage in 3 to 10 year old children