Liam O'Mahony

In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings

The enteric flora comprises approximately 95% of the total number of cells in the human body and can elicit immune responses while protecting against microbial pathogens. However, the resident bacterial flora of the gastrointestinal tract may also be implicated in the pathogenesis of diseases such as inflammatory bowel disease (ulcerative colitis and Crohn disease). The objectives of the Probiotic Research Group based at University College Cork were to isolate and identify lactic acid bacteria exhibiting beneficial probiotic traits, such as bile tolerance in the absence of deconjugation activity, acid resistance, adherence to host epithelial tissue, and in vitro antagonism of pathogenic microorganisms or those suspected of promoting inflammation. To isolate potentially effective probiotic bacteria, we screened the microbial population adhering to surgically resected segments of the gastrointestinal tract (the environment in which they may subsequently be reintroduced and required to function). In total, 1500 bacterial strains from resected human terminal ilea were assessed. From among these organisms, Lactobacillus salivarius subsp. salivarius strain UCC118 was selected for further study. In mouse feeding trials, milk-borne L. salivarius strain UCC118 could successfully colonize the murine gastrointestinal tract. A human feeding study conducted in 80 healthy volunteers showed that yogurt can be used as a vehicle for delivery of strain UCC118 to the human gastrointestinal tract with considerable efficacy in influencing gut flora and colonization. In summary, we developed criteria for in vitro selection of probiotic bacteria that may reflect certain in vivo effects on the host such as modulation of gastrointestinal tract microflora.

Efficacy of an encapsulated probiotic Bifidobacterium infantis 35624 in women with irritable bowel syndrome.

BACKGROUND:Probiotic bacteria exhibit a variety of properties, including immunomodulatory activity, which are unique to a particular strain. Thus, not all species will necessarily have the same therapeutic potential in a particular condition. We have preliminary evidence that Bifidobacterium infantis 35624 may have utility in irritable bowel syndrome (IBS).OBJECTIVES:This study was designed to confirm the efficacy of the probiotic bacteria B. infantis 35624 in a large-scale, multicenter, clinical trial of women with IBS. A second objective of the study was to determine the optimal dosage of probiotic for administration in an encapsulated formulation.METHODS:After a 2-wk baseline, 362 primary care IBS patients, with any bowel habit subtype, were randomized to either placebo or freeze-dried, encapsulated B. infantis at a dose of 1 × 106, 1 × 108, or 1 × 1010, cfu/mL for 4 wk. IBS symptoms were monitored daily and scored on to a 6-point Likert scale with the primary outcome variable being abdominal pain or discomfort. A composite symptom score, the subjects global assessment of IBS symptom relief, and measures of quality of life (using the IBS-QOL instrument) were also recorded.RESULTS:B. infantis 35624 at a dose of 1 × 108 cfu was significantly superior to placebo and all other bifidobacterium doses for the primary efficacy variable of abdominal pain as well as the composite score and scores for bloating, bowel dysfunction, incomplete evacuation, straining, and the passage of gas at the end of the 4-wk study. The improvement in global symptom assessment exceeded placebo by more than 20% (p < 0.02). Two other doses of probiotic (1 × 106 and 1 × 1010) were not significantly different from placebo; of these, the 1 × 1010 dose was associated with significant formulation problems. No significant adverse events were recorded.CONCLUSIONS:B. infantis 35624 is a probiotic that specifically relieves many of the symptoms of IBS. At a dosage level of 1 × 108 cfu, it can be delivered by a capsule making it stable, convenient to administer, and amenable to widespread use. The lack of benefits observed with the other dosage levels of the probiotic highlight the need for clinical data in the final dosage form and dose of probiotic before these products should be used in practice.

Double blind, placebo controlled trial of two probiotic strains in interleukin 10 knockout mice and mechanistic link with cytokine balance

Background: Prophylactic efficacy against colitis following lactobacillus consumption in interleukin 10 (IL-10) knockout (KO) mice has been reported. Whether this applies equally to other probiotic strains is unknown, and the mechanism is unclear. Aims: (1) To compare the effect of feeding Lactobacillus salivarius subspecies salivarius 433118 and Bifidobacterium infantis 35624 against placebo on enterocolitis, the intestinal microflora, and (2) to compare the systemic immunological response to in vitro microbial challenge in probiotic fed and control IL-10 KO mice. Methods: Three groups of 10 IL-10 KO mice were fed fermented milk products containing Lb salivarius 433118 at 109 CFU/ml, B infantis 35624 at 108 CFU/ml, and unmodified milk, respectively, for 19 weeks. Faecal samples were taken at regular intervals to confirm gut transit, recovery of fed probiotics, and to assess the impact on the microflora. At sacrifice, the bowels were histologically scored. Cytokine production from Peyers’ patches and splenocytes was measured in vitro by ELISA. Results: Faecal recovery of probiotics was confirmed in all probiotic fed mice but not in controls. Colonic and caecal inflammatory scores were significantly decreased in both groups of probiotic fed mice (p<0.05). Proinflammatory cytokine production by Peyers’ patches and splenocytes was significantly reduced in probiotic fed animals whereas transforming growth factor β (TGF-β) levels were maintained. Conclusion: Both Lactobacillus salivarius 433118 and Bifidobacterium infantis 35624 significantly attenuate colitis in this murine model. Attenuation of colitis is associated with a reduced ability to produce Th1-type cytokines systemically and mucosally, while levels of TGF-β are maintained.

Probiotics: from myth to reality. Demonstration of functionality in animal models of disease and in human clinical trials

The enteric flora comprise approximately 95% of the total number of cells in the human body and are capable of eliciting immune responses while also protecting against microbial pathogens. However, the resident bacterial flora of the gastrointestinal tract (GIT) may also be implicated in the pathogenesis of several chronic conditions such as inflammatory bowel disease (IBD). The University College Cork-based Probiotic Research Group has successfully isolated and identified lactic acid bacteria (LAB) which exhibit beneficial probiotic traits. These characteristics include the demonstration of bile tolerance; acid resistance; adherence to host epithelial tissue; and in vitro antagonism of potentially-pathogenic micro-organisms or those which have been implicated in promoting inflammation. The primary objective of this report is to describe the strategy adopted for the selection of potentially effective probiotic bacteria. The study further describes the evaluation of two m embers of the resulting panel of micro-organisms (Lactobacillus salivarius subsp. salivarius UCC118 and Bifidobacterium longum infantis 35624) under in vitro conditions and throughout in vivo murine and human feeding trials. Specifically, an initial feeding study completed in Balb/c mice focused upon (i) effective delivery of the probiotic micro-organisms to the GIT and evaluation of the ability of the introduced strains to survive transit through, and possibly colonise, the murine GIT; (ii) accepting the complexity of the hostile GIT and faecal environments, development of a method of enumerating the introduced bacterial strains using conventional microbiological techniques; and (iii) assessment of the effects of administered bacterial strains on the numbers of specific recoverable indigenous bacteria in the murine GIT and faeces. Additional research, exploiting the availability of murine models of inflammatory bowel disease, demonstrated the beneficial effects of administering probi otic combinations of Lactobacillus salivarius UCC118 and Bifidobacterium longum infantis 35624 in prevention of illness-related weight loss. A further ethically-approved feeding trial, successfully conducted in 80 healthy volunteers, demonstrated that yoghurt can be used as a vehicle for delivery of Lactobacillus salivarius strain UCC118 to the human GIT with considerable efficacy in influencing gut flora and colonisation.

Commensal-Induced Regulatory T Cells Mediate Protection against Pathogen-Stimulated NF-κB Activation

Host defence against infection requires a range of innate and adaptive immune responses that may lead to tissue damage. Such immune-mediated pathologies can be controlled with appropriate T regulatory (Treg) activity. The aim of the present study was to determine the influence of gut microbiota composition on Treg cellular activity and NF-κB activation associated with infection. Mice consumed the commensal microbe Bifidobacterium infantis 35624 followed by infection with Salmonella typhimurium or injection with LPS. In vivo NF-κB activation was quantified using biophotonic imaging. CD4+CD25+Foxp3+ T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4+ T cells were isolated using magnetic beads for adoptive transfer to naïve animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF-κB activity that preceded a reduction in S. typhimurium numbers and murine sickness behaviour scores in B. infantis–fed mice. In addition, pro-inflammatory cytokine secretion, T cell proliferation, and dendritic cell co-stimulatory molecule expression were significantly reduced. In contrast, CD4+CD25+Foxp3+ T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4+CD25+ T cells transferred the NF-κB inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF-κB activation in vivo. These cellular interactions provide the basis for a more complete understanding of the commensal-host-pathogen trilogue that contribute to host homeostatic mechanisms underpinning protection against aberrant activation of the innate immune system in response to a translocating pathogen or systemic LPS.

Probiotic impact on microbial flora, inflammation and tumour development in IL‐10 knockout mice

The enteric bacterial flora has been implicated in the pathogenesis of enterocolitis and colon cancer in C57BL/6 IL‐10 knockout mice. Probiotic Lactobacilli modify the enteric flora and are thought to have a beneficial effect on enterocolitis. We conducted a controlled feeding trial in IL‐10 knockout mice using the probiotic Lactobacillus salivarius ssp. salivarius UCC118.

EAACI Food Allergy and Anaphylaxis Guidelines. Primary prevention of food allergy

Food allergy can have significant effects on morbidity and quality of life and can be costly in terms of medical visits and treatments. There is therefore considerable interest in generating efficient approaches that may reduce the risk of developing food allergy. This guideline has been prepared by the European Academy of Allergy and Clinical Immunologys (EAACI) Taskforce on Prevention and is part of the EAACI Guidelines for Food Allergy and Anaphylaxis. It aims to provide evidence‐based recommendations for primary prevention of food allergy. A wide range of antenatal, perinatal, neonatal, and childhood strategies were identified and their effectiveness assessed and synthesized in a systematic review.

Bifidobacterium infantis 35624 administration induces Foxp3 T regulatory cells in human peripheral blood: potential role for myeloid and plasmacytoid dendritic cells

Background Intestinal homoeostasis is dependent on immunological tolerance to the microbiota. Objective To (1) determine if a probiotic could induce Foxp3 T cells in humans; (2) to elucidate the molecular mechanisms, which are involved in the induction of Foxp3 T cells by human dendritic cells. Design Cytokine secretion and Foxp3 expression were assessed in human volunteers following Bifidobacterium infantis feeding. Monocyte-derived dendritic cells (MDDCs), myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) were incubated in vitro with B infantis and autologous lymphocytes. Transcription factor expression, costimulatory molecule expression, cytokine secretion, retinoic acid and tryptophan metabolism were analysed. Results Volunteers fed B infantis displayed a selective increase in secretion of interleukin (IL)-10 and enhanced Foxp3 expression in peripheral blood. In vitro, MDDCs, mDCs and pDCs expressed indoleamine 2,3-dioxygenase and secreted IL-10, but not IL-12p70, in response to B infantis. MDDC and mDC IL-10 secretion was Toll-like receptor (TLR)-2/6 dependent, while pDC IL-10 secretion was TLR-9 dependent. In addition, MDDCs and mDCs expressed RALDH2, which was TLR-2 and DC-SIGN dependent. B infantis-stimulated MDDCs, mDCs and pDCs induced T cell Foxp3 expression. TLR-2, DC-SIGN and retinoic acid were required for MDDC and mDC induction of Foxp3 T cells, while pDCs required indoleamine 2,3-dioxygenase. Conclusions B infantis administration to humans selectively promotes immunoregulatory responses, suggesting that this microbe may have therapeutic utility in patients with inflammatory disease. Cross-talk between multiple pattern-recognition receptors and metabolic pathways determines the innate and subsequent T regulatory cell response to B infantis. These findings link nutrition, microbiota and the induction of tolerance within the gastrointestinal mucosa.

Metabolic activity of the enteric microbiota influences the fatty acid composition of murine and porcine liver and adipose tissues

BACKGROUND Recent reports suggest that the metabolic activity of the gut microbiota may contribute to the pathogenesis of obesity and hepatic steatosis. OBJECTIVE The objective was to determine whether the fat composition of host tissues might be influenced by oral administration of commensal bifidobacteria previously shown by us to produce bioactive isomers of conjugated linoleic acid (CLA). DESIGN Murine trials were conducted in which linoleic acid-supplemented diets were fed with or without Bifidobacterium breve NCIMB 702258 (daily dose of 10(9) microorganisms) to healthy BALB/c mice and to severe combined immunodeficient mice for 8-10 wk. To ensure that the observations were not peculiar to mice, a similar trial was conducted in weanling pigs over 21 d. Tissue fatty acid composition was assessed by gas-liquid chromatography. RESULTS In comparison with controls, there was an increase in cis-9, trans-11 CLA in the livers of the mice and pigs after feeding with linoleic acid in combination with B. breve NCIMB 702258 (P < 0.05). In addition, an altered profile of polyunsaturated fatty acid composition was observed, including higher concentrations of the omega-3 (n-3) fatty acids eicosapentaenoic acid and docosahexaenoic acid in adipose tissue (P < 0.05). These changes were associated with reductions in the proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma (P < 0.05). CONCLUSIONS These results are consistent with the concept that the metabolome is a composite of host and microbe metabolic activity and that the influence of the microbiota on host fatty acid composition can be manipulated by oral administration of CLA-producing microorganisms.

Bacterial strain-specific induction of Foxp3+ T regulatory cells is protective in murine allergy models

Background The incidence of atopic disease has increased dramatically during recent decades and the potential immunoregulatory influence of the microbiota in these individuals is under investigation.