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Vancomycin, metronidazole, and the bacteriocin lacticin 3147 are active against a wide range of bacterial species, including Clostridium difficile. We demonstrate that, in a human distal colon model, the addition of each of the three antimicrobials resulted in a significant decrease in numbers of C. difficile. However, their therapeutic use in the gastrointestinal tract may be compromised by their broad spectrum of activity, which would be expected to significantly impact on other members of the human gut microbiota. We used highthroughput pyrosequencing to compare the effect of each antimicrobial on the composition of the microbiota. All three treatments resulted in a decrease in the proportion of sequences assigned to the phyla Firmicutes and Bacteroidetes, with a corresponding increase in those assigned to members of the Proteobacteria. One possible means of avoiding such \"collateral damage\" would involve the application of a narrow-spectrum antimicrobial with specific anti-C. difficile activity. We tested this hypothesis using thuricin CD, a narrowspectrum bacteriocin produced by Bacillus thuringiensis, which is active against C. difficile. The results demonstrated that this bacteriocin was equally effective at killing C. difficile in the distal colon model but had no significant impact on the composition of the microbiota. This offers the possibility of developing a targeted approach to eliminating C. difficile in the colon, without collateral damage.

Comorbidity with Clostridium difficile may cause diagnostic delay in newly presenting inflammatory bowel disease (IBD) patients, trigger relapse in established disease, confound therapies, and serve as an indicator of an underlying defect in innate immunity. Retrospective analyses have suggested community acquisition; to address this we conducted a prospective analysis of C. difficile carriage in IBD patients using molecular methods specifically in an outpatient setting. Recruited participants had long-standing diagnoses of ulcerative colitis (n = 64) and Crohn's disease (n = 58), were in clinical remission, and had no recent exposure to antibiotics, corticosteroids, immunomodulatory drugs or recent hospitalization. Isolates were cultured from stools and confirmed by 16S sequencing. The antibiotic susceptibilities of the isolates were tested followed by further strain characterization by toxinotyping, ribotyping, and pulsed-field gel electrophoresis (PFGE). The frequency of toxigenic C. difficile was higher in IBD patients than in healthy volunteers at 8.2 and 1.0%, respectively (P = 0.02 Fisher's exact test). All strains belonged to toxinotype 0 with rare subtypes of this group noted in five isolates and represented by an altered repressor genotype. Patients harbored a diverse range of toxigenic ribotype groups, including those previously associated with C. difficile-associated disease (CDAD) (R015, R005, and R020) and the rarer types R062, R050, and R003. Interestingly, common nosocomial groups were not identified. The considerable nonclonal distribution of distinct strains was further demonstrated by PFGE genomic fingerprinting. None of the study subjects experienced a clinical episode of CDAD during a 6-month period of follow-up. Detection of C. difficile is increased in IBD outpatients in remission, and strain diversity is consistent with community acquisition from a multitude of sources.

Recently, we reported that administration of Bifidobacteria resulted in increased concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in murine adipose tissue [1]. The objective of this study was to assess the impact of co-administration of Bifidobacterium breve NCIMB 702258 and the substrate for EPA, α-linolenic acid, on host fatty acid composition. α-Linolenic acid-supplemented diets (1%, wt/wt) were fed to mice (n = 8), with or without B. breve NCIMB 702258 (daily dose of 10⁹ microorganisms) for 8 weeks. Two further groups received either supplement of B. breve alone or unsupplemented diet. Tissue fatty acid composition was assessed by gas liquid chromatography. Dietary supplementation of α-linolenic acid resulted in higher (P < 0.05) α-linolenic acid and EPA concentrations in liver and adipose tissue and lower (P < 0.05) arachidonic acid in liver, adipose tissue and brain compared with mice that did not receive α-linolenic acid. Supplementation with B. breve NCIMB 702258 in combination with α-linolenic acid resulted in elevated (P < 0.05) liver EPA concentrations compared with α-linolenic acid supplementation alone. Furthermore, the former group had higher (P < 0.05) DHA in brain compared with the latter group. These results suggest a role for interactions between fatty acids and commensals in the gastrointestinal tract. This interaction between administered microbes and fatty acids could result in a highly effective nutritional approach to the therapy of a variety of inflammatory and neurodegenerative conditions.

The aim of this study was to compare the impact of dietary supplementation with a Bifidobacterium breve strain together with linoleic acid & α-linolenic acid, for 7 weeks, on colonic sensitivity and fatty acid metabolism in rats. Maternally separated and non-maternally separated Sprague Dawley rats (n = 15) were orally gavaged with either B. breve DPC6330 (10(9) microorganisms/day) alone or in combination with 0.5% (w/w) linoleic acid & 0.5% (w/w) α-linolenic acid, daily for 7 weeks and compared with trehalose and bovine serum albumin. Tissue fatty acid composition was assessed by gas-liquid chromatography and visceral hypersensitivity was assessed by colorectal distension. Significant differences in the fatty acid profiles of the non-separated controls and maternally separated controls were observed for α-linolenic acid and arachidonic acid in the liver, oleic acid and eicosenoic acid (c11) in adipose tissue, and for palmitoleic acid and docosahexaenoic acid in serum (p<0.05). Administration of B. breve DPC6330 to MS rats significantly increased palmitoleic acid, arachidonic acid and docosahexaenoic acid in the liver, eicosenoic acid (c11) in adipose tissue and palmitoleic acid in the prefrontal cortex (p<0.05), whereas feeding B. breve DPC6330 to non separated rats significantly increased eicosapentaenoic acid and docosapentaenoic acid in serum (p<0.05) compared with the NS un-supplemented controls. Administration of B. breve DPC6330 in combination with linoleic acid and α-linolenic acid to maternally separated rats significantly increased docosapentaenoic acid in the serum (p<0.01) and α-linolenic acid in adipose tissue (p<0.001), whereas feeding B. breve DPC6330 with fatty acid supplementation to non-separated rats significantly increased liver and serum docosapentaenoic acid (p<0.05), and α-linolenic acid in adipose tissue (p<0.001). B. breve DPC6330 influenced host fatty acid metabolism. Administration of B. breve DPC6330 to maternally separated rats significantly modified the palmitoleic acid, arachidonic acid and docosahexaenoic acid contents in tissues. The effect was not observed in non-separated animals.

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 members 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 probiotic 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.

Background Bifidobacteria and lactobacilli are among the early and important colonizers of the gastrointestinal tract and are generally considered to be part of a normal, healthy microbiota. It is believed that specific strains within the microbiota can influence host immune-reactivity and may play a role in protection from infection and aberrant inflammatory activity. One such strain, Bifidobacterium animalis AHC7, has been previously shown to protect against Salmonella typhimurium infection in mice and helps resolve acute idiopathic diarrhea in dogs. The aim of this study was to investigate the potential molecular and cellular mechanisms underpinning the Bifidobacterium animalis AHC7 protective effect. Results Following 4 hours of infection with Salmonella typhimurium , NF-κB activation was significantly elevated in vivo in placebo and Enterococcus faecium -fed animals while Bifidobacterium animalis AHC7 consumption significantly attenuated the NF-κB response. In vitro anti-CD3/CD28 stimulated Peyer's patch cells secreted significantly less TNF-α and IFN-γ following Bifidobacterium animalis AHC7 consumption. Stimulated cells released more IL-12p70 but this difference did not reach statistical significance. No alteration in mucosal IL-6, IL-10 or MCP-1 levels were observed. No statistically significant change in the cytokine profile of mesenteric lymph node cells was noted. In vitro , Bifidobacterium animalis AHC7 was bound by dendritic cells and induced secretion of both IL-10 and IL-12p70. In addition, co-culture of CD4+ T cells with Bifidobacterium animalis AHC7-stimulated dendritic cells resulted in a significant increase in CD25+Foxp3+ T cell numbers. Conclusion Bifidobacterium animalis AHC7 exerts an anti-inflammatory effect via the attenuation of pro-inflammatory transcription factor activation in response to an infectious insult associated with modulation of pro-inflammatory cytokine production within the mucosa. The cellular mechanism underpinning Bifidobacterium animalis AHC7 mediated attenuation of NF-κB activation may include recognition of the bacterium by dendritic cells and induction of CD25+Foxp3+ T cells.

BackgroundIntestinal homoeostasis is dependent on immunological tolerance to the microbiota.ObjectiveTo (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.DesignCytokine 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.ResultsVolunteers 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.ConclusionsB 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.

Proteolytic degradation of the extracellular matrix, a feature of mucosal homeostasis and tissue renewal, also contributes to the complications of intestinal inflammation. Whether this proteolytic activity is entirely host-derived, or, in part, produced by the gut microbiota, is unknown.MethodsWe screened the bacterial colonies for gelatinolytic activity from fecal samples of 20 healthy controls, 23 patients with ulcerative colitis, and 18 with Crohn's disease (CD). In addition, the genes encoding metalloproteases were detected by conventional or real-time polymerase chain reaction (PCR).ResultsGelatinolytic activity was found in approximately one-quarter of samples regardless of the presence of inflammation and without any attempt to enhance the sensitivity of the culture-based screen. This was associated with a diversity of bacteria, particularly in CD, but was predominantly linked with Clostridium perfringens. Culture supernatants from C. perfringens degraded gelatin, azocoll, type I collagen, and basement membrane type IV collagen, but different isolates varied in the degree of proteolytic activity. Results were confirmed by detection of the C. perfringens colA gene (encoding collagenase) in fecal DNA, again regardless of the presence or absence of inflammation. However, the biologic significance and potential implications of microbial-derived proteolytic activity were confirmed by reduced transepithelial resistance (TER) after exposure of rat distal colon to culture supernatants of C. perfringens in Ussing chambers.ConclusionsThe study shows that microbial-derived proteolytic activity has the capacity to contribute to mucosal homeostasis and may participate in the pathogenesis of inflammatory bowel disease. (Inflamm Bowel Dis 2010)

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- Kappa B 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- Kappa B activation was quantified using biophotonic imaging. CD4 super(+)CD25 super(+)Foxp3 super(+) T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4 super(+) T cells were isolated using magnetic beads for adoptive transfer to naive animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF- Kappa B 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 super(+)CD25 super(+)Foxp3 super(+) T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4 super(+)CD25 super(+) T cells transferred the NF- Kappa B inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF- Kappa B 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. Author Summary The normal response to infection is rapid and effective clearance of pathogenic microbes. However, this immune response may occasionally cause collateral inflammatory damage to host tissue and in severe cases, such as systemic sepsis, results in organ failure. Various cellular mechanisms, including regulatory T cells, protect against aggressive immune responses. However, environmental agents which promote regulatory T cells are not well understood. We and others have previously shown that non-pathogenic or commensal micro-organisms can protect the host from aberrant pro-inflammatory activity within the gut, but the influence of these microbes on regulatory T cells in the context of systemic infection has not been examined. In this study, we demonstrate that consumption of a single commensal bacterium induces regulatory T cells in vivo which protect the host from pathogen-induced inflammatory responses by limiting activation of the pro-inflammatory transcription factor NF- Kappa B via the toll-like receptor 4 (TLR-4) pathway. This report conclusively demonstrates a cellular and molecular basis for the commensal-host-pathogen trilogue resulting in enhanced protection from systemic infection whilst limiting pro-inflammatory damage mediated by activation of the innate immune system.

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). 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. 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 x 10(6), 1 x 10(8), or 1 x 10(10), 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 subject's global assessment of IBS symptom relief, and measures of quality of life (using the IBS-QOL instrument) were also recorded. B. infantis 35624 at a dose of 1 x 10(8) 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 x 10(6) and 1 x 10(10)) were not significantly different from placebo; of these, the 1 x 10(10) dose was associated with significant formulation problems. No significant adverse events were recorded. B. infantis 35624 is a probiotic that specifically relieves many of the symptoms of IBS. At a dosage level of 1 x 10(8) 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.