Intranasal Bifidobacterium longum protects against viral-induced lung inflammation and injury in a murine model of lethal influenza infection.

BACKGROUND: Prophylactic strategies are urgently needed for prevention of severe inflammatory responses to respiratory viral infections. Bacterial-host interactions may modify the immune response to viral infections. METHODS: We examined the contribution of Intranasal administration of two different Bifidobacterium longum strains or its isolated cell wall in controlling viral induced inflammation using a murine model of influenza infection. We monitored mortality and morbidity over a 10-day period and viral load, differential broncho alveolar lavage (BAL) fluid inflammatory cell counts, Lung tissue histology, BAL and serum cytokines, markers of vascular damage and cell death were quantified. FINDINGS: Intranasal administration of Bifidobacterium longum35624® or its isolated cell wall prior to virus inoculation significantly reduced viral load within the lungs and significantly improved survival. Reduced viral load was associated with reduced lung injury as suggested by cell death and vascular leakage markers, a shift from neutrophil to macrophage recruitment, reduced inflammatory cytokine levels (including IL-6), reduced type 1 and 2 interferon levels, but increased levels of interferon-λ and surfactant protein D. These protective effects were maintained when the bifidobacterial cell wall preparation was administered 24 h after viral inoculation. The protective effects were also observed for the Bifidobacterium longumPB-VIR™ strain. INTERPRETATION: Exposure to these bifidobacterial strains protect against the inflammatory sequelae and damage associated with uncontrolled viral replication within the lung. FUNDING: This work has been funded, in part, by a research grant from GlaxoSmithKline, PrecisionBiotics Group Ltd., Swiss National Science Foundation grants (project numbers CRSII3_154488, 310030_144219, 310030_127356 and 310030_144219) and Christine Kühne - Center for Allergy Research and Education (CK-CARE).

Lactobacillus gasseri APC 678 Reduces Shedding of the Pathogen Clostridium difficile in a Murine Model.

Clostridium difficile is a common cause of health-care acquired diarrhea, resulting in a spectrum of disease from mild diarrhea to life-threatening illness. Sixty Lactobacillus strains were screened for anti-C. difficile activity using a co-culture method. Based on their ability to inhibit C. difficile, L. gasseri APC 678 and L. rhamnosus DPC 6111 were selected for study in a murine model of C. difficile infection. L. gasseri ATCC 33323, was included as a control. It was established that, relative to control mice not fed Lactobacillus, feeding with L. gasseri APC 678 resulted in a significant reduction by day 7 (8-fold, p = 0.017) of viable C. difficile VPI 10463 in the feces of mice. In contrast, neither L. rhamnosus DPC 6111 nor L. gasseri ATCC 33323 significantly reduced fecal C. difficile shedding. Sequencing of the cecal microbiota showed that in mice fed L. gasseri APC 678 there was a significant increase in bacterial diversity across a number of indices when compared to the control or other Lactobacillus-fed groups. There was no significant change in the relative abundance of Firmicutes or Bacteroidetes in the group fed L. gasseri APC 678 relative to the control, while the groups fed L. rhamnosus DPC 6111 or L. gasseri ATCC 33323 showed a significant decrease in the relative abundance of Firmicutes (p = 0.002 and p = 0.019, respectively) and a significant increase in Bacteroidetes (p = 0.002 and p = 0.023, respectively). These results highlight the potential of L. gasseri APC 678 as a live therapeutic agent to target C. difficile infection.

Bioavailability of the anti-clostridial bacteriocin thuricin CD in gastrointestinal tract.

Thuricin CD is a two component narrow spectrum bacteriocin comprising two peptides with targeted activity against Clostridium difficile. This study examined the bioavailability of thuricin with a view to developing it as an effective antimicrobial against intestinal infection. One of the peptides, Trn-ß, was found to be degraded by the gastric enzymes pepsin and α-chymotrypsin both in vitro and in vivo, whereas Trn-α was resistant to digestion by these enzymes and hence was detected in the intestinal porcine digesta following oral ingestion by pigs. In order to determine if spores of the producing organism Bacillus thuringiensis DPC 6431 could be used to deliver the bacteriocin to the gut, spores were fed to 30 mice (approx. 10(8)-2×10(8) per animal) and their germination, growth and production of thuricin in the gastrointestinal tract (GIT) of the animals was monitored. Almost 99 % of the spores delivered to the GIT were excreted in the first 24 h and neither Trn-α nor Trn-ß was detected in the gut or faecal samples of the test mice, indicating that ingestion of B. thuringiensis spores may not be a suitable vehicle for the delivery of thuricin CD. When thuricin CD was delivered rectally to mice (n = 40) and C. difficile shedding monitored at 1, 6, 12 and 24 h post-treatment, there was a >95 % (>1.5 log units) reduction of C. difficile 027 in the colon contents of infected mice (n = 10) 1 h post-treatment compared with the control group (n = 10; P<0.001). Furthermore, 6 h post-treatment there was a further 1.5 log reduction in C. difficile numbers (n = 10) relative to the control group (n = 10; P<0.05). These results would suggest that rectal administration of thuricin may be a promising mode of delivery of thuricin CD to the colon.

Bifidobacterium infantis 35624 modulates host inflammatory processes beyond the gut.

Certain therapeutic microbes, including Bifidobacteria infantis (B. infantis) 35624 exert beneficial immunoregulatory effects by mimicking commensal-immune interactions; however, the value of these effects in patients with non-gastrointestinal inflammatory conditions remains unclear. In this study, we assessed the impact of oral administration of B. infantis 35624, for 6‒8 weeks on inflammatory biomarker and plasma cytokine levels in patients with ulcerative colitis (UC) (n = 22), chronic fatigue syndrome (CFS) (n = 48) and psoriasis (n = 26) in three separate randomized, double-blind, placebo-controlled interventions. Additionally, the effect of B. infantis 35624 on immunological biomarkers in healthy subjects (n = 22) was assessed. At baseline, both gastrointestinal (UC) and non-gastrointestinal (CFS and psoriasis) patients had significantly increased plasma levels of C-reactive protein (CRP) and the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) compared with healthy volunteers. B. infantis 35624 feeding resulted in reduced plasma CRP levels in all three inflammatory disorders compared with placebo. Interestingly, plasma TNF-α was reduced in CFS and psoriasis while IL-6 was reduced in UC and CFS. Furthermore, in healthy subjects, LPS-stimulated TNF-α and IL-6 secretion by peripheral blood mononuclear cells (PBMCs) was significantly reduced in the B. infantis 35624-treated groups compared with placebo following eight weeks of feeding. These results demonstrate the ability of this microbe to reduce systemic pro-inflammatory biomarkers in both gastrointestinal and non-gastrointestinal conditions. In conclusion, these data show that the immunomodulatory effects of the microbiota in humans are not limited to the mucosal immune system but extend to the systemic immune system.

Bifidobacterium breve with α-linolenic acid and linoleic acid alters fatty acid metabolism in the maternal separation model of irritable bowel syndrome.

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.

Carriage of Clostridium difficile in outpatients with irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a common, typically chronic and sometimes disabling gastrointestinal condition of uncertain aetiology. Recently, a variety of links to gastrointestinal infections have been described including the onset of IBS following exposure to enteric pathogens and an apparent predisposition to gastrointestinal infection. The prevalence of Clostridium difficile in a population of IBS outpatients (n = 87) in the absence of established risk factors for the acquisition of C. difficile infection was examined. Overall, 5.7 % of patients (n = 5) carried culturable C. difficile and 4.6 % (n = 4) of isolates were toxigenic, belonging to toxinotype group 0, compared with 1.1 % (n = 1) for the healthy control group (n = 88). These isolates were members of toxigenic PCR ribotype groups 005 and 050 (IBS group) and 062 (control group) and were identified further as three individual strains by PFGE. Although no significant difference was observed between IBS patients and healthy volunteers, these findings support the concept that a subpopulation of IBS patients may be susceptible to gastrointestinal infection.

Contrasting effects of Bifidobacterium breve NCIMB 702258 and Bifidobacterium breve DPC 6330 on the composition of murine brain fatty acids and gut microbiota.

BACKGROUND: We previously showed that microbial metabolism in the gut influences the composition of bioactive fatty acids in host adipose tissue. OBJECTIVE: This study compared the effect of dietary supplementation for 8 wk with human-derived Bifidobacterium breve strains on fat distribution and composition and the composition of the gut microbiota in mice. METHODS: C57BL/6 mice (n = 8 per group) received B. breve DPC 6330 or B. breve NCIMB 702258 (10(9) microorganisms) daily for 8 wk or no supplement (controls). Tissue fatty acid composition was assessed by gas-liquid chromatography while 16S rRNA pyrosequencing was used to investigate microbiota composition. RESULTS: Visceral fat mass and brain stearic acid, arachidonic acid, and DHA were higher in mice supplemented with B. breve NCIMB 702258 than in mice in the other 2 groups (P < 0.05). In addition, both B. breve DPC 6330 and B. breve NCIMB 702258 supplementation resulted in higher propionate concentrations in the cecum than did no supplementation (P < 0.05). Compositional sequencing of the gut microbiota showed a tendency for greater proportions of Clostridiaceae (25%, 12%, and 18%; P = 0.08) and lower proportions of Eubacteriaceae (3%, 12%, and 13%; P = 0.06) in mice supplemented with B. breve DPC 6330 than in mice supplemented with B. breve NCIMB 702258 and unsupplemented controls, respectively. CONCLUSION: The response of fatty acid metabolism to administration of bifidobacteria is strain-dependent, and strain-strain differences are important factors that influence modulation of the gut microbial community by ingested microorganisms.

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.

Degradation of the extracellular matrix components by bacterial-derived metalloproteases: implications for inflammatory bowel diseases.

BACKGROUND: 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. METHODS: We 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). RESULTS: Gelatinolytic 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. CONCLUSIONS: The 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.

Effect of broad- and narrow-spectrum antimicrobials on Clostridium difficile and microbial diversity in a model of the distal colon.

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 high-throughput 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 narrow-spectrum 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.

Bifidobacterium animalis AHC7 protects against pathogen-induced NF-κB activation in vivo.

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.

Impact of administered bifidobacterium on murine host fatty acid composition.

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, alpha-linolenic acid, on host fatty acid composition. alpha-Linolenic acid-supplemented diets (1%, wt/wt) were fed to mice (n = 8), with or without B. breve NCIMB 702258 (daily dose of 10(9) 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 alpha-linolenic acid resulted in higher (P < 0.05) alpha-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 alpha-linolenic acid. Supplementation with B. breve NCIMB 702258 in combination with alpha-linolenic acid resulted in elevated (P < 0.05) liver EPA concentrations compared with alpha-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.

Clinical benefits of probiotic canine-derived Bifidobacterium animalis strain AHC7 in dogs with acute idiopathic diarrhea.

This study evaluated the effect of supplementation with canine-derived probiotic Bifidobacterium animalis strain AHC7 (lams Prostora, Procter & Gamble Pet Care) on the resolution rate of acute idiopathic diarrhea in dogs randomly assigned to receive a placebo (n=18) or the probiotic (n=13). Nutritional management with the probiotic fed at 2 x 10(10) CFU/day significantly reduced the time to resolution (3.9 +/- 2.3 versus 6.6 +/- 2.7 days; P < .01) and reduced the percentage of dogs that were administered metronidazole (38.5% versus 50.0%) compared with placebo. Probiotic B. animalis AHC7 may provide veterinarians another tool for management of acute diarrhea in dogs.

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.

The vexed relationship between Clostridium difficile and inflammatory bowel disease: an assessment of carriage in an outpatient setting among patients in remission.

OBJECTIVES: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: Detection of C. difficile is increased in IBD outpatients in remission, and strain diversity is consistent with community acquisition from a multitude of sources.

Mucosal cytokine imbalance in irritable bowel syndrome.

OBJECTIVE: To systematically examine mucosal biopsies for differences in cytokine gene expression and protein secretion. MATERIAL AND METHODS: The study included 59 females with irritable bowel syndrome (IBS) and 39, otherwise healthy, female volunteers presenting for colonoscopy. Colonic biopsies from subsets were studied by microarray analysis (IBS, n=9; controls, n=8), quantitative reverse transcription-polymerase chain reaction (qRT-PCR) (IBS, n=22; controls, n=21), and ex vivo biopsy culture (IBS, n=28, controls, n=10). Biopsies from patients with active colitis were used as inflammatory disease controls. RESULTS: While gene array analysis revealed extensive overlapping between controls and IBS patients, reduced expression of genes linked to chemokine function was evident among the IBS patients alone. Differential expression was confirmed by qRT-PCR or ex vivo biopsy culture for 5 out of 6 selected genes. Reduced secretion of chemokines (IL-8, CXCL-9 and MCP-1) but not pro-inflammatory cytokines (TNF-alpha, IL-6 and IL-1beta) was established on the basis of the ex vivo biopsy cultures. These findings were in marked contrast to the IBD patients who demonstrated increased production of both chemokines and pro-inflammatory cytokines. CONCLUSIONS: Despite the expected heterogeneity of the disorder, differences in mucosal chemokine signalling were evident in this cross-sectional study of IBS patients at the level of both gene expression and protein secretion, with IBS patients demonstrating a consistent deficit in the expression and secretion of chemokines known to play a critical role in mucosal defence.

Commensal-induced regulatory T cells mediate protection against pathogen-stimulated NF-kappaB 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-kappaB 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-kappaB 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-kappaB 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-kappaB inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF-kappaB 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.

Antimicrobial activity of lacticin 3,147 against clinical Clostridium difficile strains.

Clostridium difficile-associated diarrhoea (CDAD) is the most common hospital-acquired diarrhoea, and is a major type of gastroenteritis infection in nursing homes and facilities for the elderly. In this study the antimicrobial activity of the two-component lantibiotic, lacticin 3,147, against a range of genetically distinct C. difficile isolates was studied. The bacteriocin exhibited an MIC(50) of 3.6 microg ml(-1) for 10 genetically distinct C. difficile strains isolated from healthy subjects, inflammatory bowel disease patients and culture collection strains. In time-kill studies, 10(6) c.f.u. ml(-1) C. difficile ATCC 42,593 and CDAD isolate DPC 6,220 were killed within 120 or 20 min incubation, respectively, at a concentration of 6 microg lacticin ml(-1). Interestingly, addition of lacticin 3,147 to exponentially growing cells of C. difficile ATCC 43,593 caused rapid lysis of the cells after an initial lag phase, as measured by the concomitant release of the intracellular enzyme, acetate kinase. The addition of a food-grade, milk-based lacticin containing powder to faecal fermentation demonstrated that lacticin is effective in completely eliminating 10(6) c.f.u. C. difficile ml(-1) from a model faecal environment within 30 min when present at concentrations as low as 18 microg ml(-1). While other culturable microflora such as total anaerobes, bacteroides, total non-spore-forming anaerobes and total Gram-negative anaerobes were unaffected, populations of lactobacilli and bifidobacteria were reduced by 3 log cycles at bacteriocin levels sufficient to eliminate over 10(6) C. difficile. In light of these findings, the potential of lacticin 3,147 for treatment of CDAD is discussed.

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 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. RESULTS: 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. CONCLUSIONS: 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.

Lactobacillus and bifidobacterium in irritable bowel syndrome: symptom responses and relationship to cytokine profiles.

BACKGROUND & AIMS: The aim of this study was to compare the response of symptoms and cytokine ratios in irritable bowel syndrome (IBS) with ingestion of probiotic preparations containing a lactobacillus or bifidobacterium strain. METHODS: Seventy-seven subjects with IBS were randomized to receive either Lactobacillus salivarius UCC4331 or Bifidobacterium infantis 35624, each in a dose of 1 x 10 10 live bacterial cells in a malted milk drink, or the malted milk drink alone as placebo for 8 weeks. The cardinal symptoms of IBS were recorded on a daily basis and assessed each week. Quality of life assessment, stool microbiologic studies, and blood sampling for estimation of peripheral blood mononuclear cell release of the cytokines interleukin (IL)-10 and IL-12 were performed at the beginning and at the end of the treatment phase. RESULTS: For all symptoms, with the exception of bowel movement frequency and consistency, those randomized to B infantis 35624 experienced a greater reduction in symptom scores; composite and individual scores for abdominal pain/discomfort, bloating/distention, and bowel movement difficulty were significantly lower than for placebo for those randomized to B infantis 35624 for most weeks of the treatment phase. At baseline, patients with IBS demonstrated an abnormal IL-10/IL-12 ratio, indicative of a proinflammatory, Th-1 state. This ratio was normalized by B infantis 35624 feeding alone. CONCLUSIONS: B infantis 35624 alleviates symptoms in IBS; this symptomatic response was associated with normalization of the ratio of an anti-inflammatory to a proinflammatory cytokine, suggesting an immune-modulating role for this organism, in this disorder.