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The gut microbiota has been shown to play diverse roles in human health and disease although the underlying mechanisms have not yet been fully elucidated. Large cohort studies can provide further understanding into inter-individual differences, with more precise characterization of the pathways by which the gut microbiota influences human physiology and disease processes. Here, we aimed to profile the stool microbiome of children and adults from two population-based cohort studies, comprising 2,111 children in the age-range of 9 to 12 years (the Generation R Study) and 1,427 adult individuals in the range of 46 to 88 years of age (the Rotterdam Study). For the two cohorts, 16S rRNA gene profile datasets derived from the Dutch population were generated. The comparison of the two cohorts showed that children had significantly lower gut microbiome diversity. Furthermore, we observed higher relative abundances of genus Bacteroides in children and higher relative abundances of genus Blautia in adults. Predicted functional metagenome analysis showed an overrepresentation of the glycan degradation pathways, riboflavin (vitamin B2), pyridoxine (vitamin B6) and folate (vitamin B9) biosynthesis pathways in children. In contrast, the gut microbiome of adults showed higher abundances of carbohydrate metabolism pathways, beta-lactam resistance, thiamine (vitamin B1) and pantothenic (vitamin B5) biosynthesis pathways. A predominance of catabolic pathways in children (valine, leucine and isoleucine degradation) as compared to biosynthetic pathways in adults (valine, leucine and isoleucine biosynthesis) suggests a functional microbiome switch to the latter in adult individuals. Overall, we identified compositional and functional differences in gut microbiome between children and adults in a population-based setting. These microbiome profiles can serve as reference for future studies on specific human disease susceptibility in childhood, adulthood and specific diseased populations.

Objectives Many countries use faecal immunochemical testing (FIT) to screen for colorectal cancer. There is increasing evidence that faecal microbiota play a crucial role in colorectal cancer carcinogenesis. We assessed the possibility of measuring faecal microbial features in FIT as potential future biomarkers in colorectal cancer screening. Methods Bacterial stability over time and the possibility of bacterial contamination were evaluated using quantitative polymerase chain reaction analysis. Positive FIT samples (n = 200) of an average-risk screening cohort were subsequently analysed for universal 16S, and bacteria. Escherichia coli (E. coli), Fusobacterium nucleatum (F. nucleatum), Bacteroidetes and Faecalibacterium prausnitzii (F. prausnitzii) by qPCR. The results were compared with colonoscopy findings. Results Faecal microbiota in FIT were stably measured up to six days for E. coli (p = 0.53), F. nucleatum (p = 0.30), Bacteroidetes (p = 0.05) and F. prausnitzii (p = 0.62). Overall presence of bacterial contamination in FIT controls was low. Total bacterial load (i.e. 16S) was significantly higher in patients with colorectal cancer and high-grade dysplasia (p = 0.006). For the individual bacteria tested, no association was found with colonic lesions. Conclusions These results show that the faecal microbial content can be measured in FIT samples and remains stable for six days. Total bacterial load was higher in colorectal cancer and high-grade dysplasia. These results pave the way for further research to determine the potential role of microbiota assessment in FIT screening.

Background Homeostasis of the gastrointestinal tract depends on a healthy bacterial microbiota, with alterations in microbiota composition suggested to contribute to diseases. To unravel bacterial contribution to disease pathology, a thorough understanding of the microbiota of the complete gastrointestinal tract is essential. To date, most microbial analyses have either focused on faecal samples, or on the microbial constitution of one gastrointestinal location instead of different locations within one individual. Objective We aimed to analyse the mucosal microbiome along the entire gastrointestinal tract within the same individuals. Methods Mucosal biopsies were taken from nine different sites in 14 individuals undergoing antegrade and subsequent retrograde double-balloon enteroscopy. The bacterial composition was characterised using 16 S rRNA sequencing with Illumina Miseq. Results At double-balloon enteroscopy, one individual had a caecal adenocarcinoma and one individual had Peutz-Jeghers polyps. The composition of the microbiota distinctively changed along the gastrointestinal tract with larger bacterial load, diversity and abundance of Firmicutes and Bacteroidetes in the lower gastrointestinal tract than the upper gastrointestinal tract, which was predominated by Proteobacteria and Firmicutes. Conclusions We show that gastrointestinal location is a larger determinant of mucosal microbial diversity than inter-person differences. These data provide a baseline for further studies investigating gastrointestinal microbiota-related disease.

Background The concurrent occurrence of gut and skin diseases has led to the postulation of the existence of a gut-skin axis in which gut microbiota is thought to contribute to extra-intestinal skin manifestations. A dysbiotic skin microbiome has been demonstrated in the chronic inflammatory skin disease hidradenitis suppurativa (HS), but it remains unknown whether microbial disturbances of the gut are engaged in the complex pathogenesis. This study investigates the fecal microbiome in HS patients and healthy controls and explores the possibility of a gut-skin axis in HS. Methods A total of 17 HS patients and 20 healthy controls provided a stool sample for 16S ribosomal RNA (rRNA) gene sequencing. A subset of axillary skin samples was available for concurrent microbial analysis of HS lesions (n=6), paired non-lesions (n=3) and healthy skin of controls (n=7). No oral antibiotics and no topical antibiotics/steroids were used in respectively eight weeks and seven days prior to sample collection. Results The majority of 16S rRNA gene sequences at both the fecal and axillary skin sites were assigned to phyla Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria. Based on the overall bacterial community, two distinct clusters could be distinguished between the gut and the skin using principal coordinates analysis (PCoA). Furthermore, significant differences in bacterial composition were observed in fecal samples of HS patients when compared to healthy controls. However, we did not detect altered microbial signatures in the skin samples taken from HS lesions, non-lesions, and healthy controls. Conclusions These findings indicate the presence of a distinct fecal microbiome in HS patients compared to healthy controls, suggesting the involvement of a gut-skin axis in the etiology of HS. Dysbiosis of the cutaneous microbiome in HS could not be confirmed here, but larger studies are warranted to further elucidate the role of microbiota as a potential part of the gut-skin axis in HS.

Pancreatic cancer with its associated infaust prognosis urgently needs better early detection of disease, especially for screening of high risk individuals. Despite early promise and theoretical considerations, however, KRAS mutional analysis of cfDNA seems not a way forward here. A more multifaceted panel of mutational spectrum biomarkers study (KRAS, TP53, SMAD4, P16, EGFR GNAS, MENN1, DAXX, VHL and in combination of pancreatic cancer specific STR markers) along with epigenetic alterations in cfDNA in pancreatic cancer patients may still provide a successful prognostic and diagnostic avenue, but generally speaking cfDNA does not appear very promising direction. Hence focus should now be directed to alternative methodology.

Background and Aims: Psoriasis and hidradenitis suppurativa [HS] co-occur more often with inflammatory bowel disease [IBD] than expected, due to shared pathogenic and genetic features. It is known that IBD patients harbour an altered intestinal microbiome characterised by a depletion of Faecalibacterium prausnitzii and increase of Escherichia coli . At present, it is unclear whether a similar intestinal microbiome trend can be identified in IBD-associated skin disorders. We therefore investigated the F. prausnitzii and E. coli abundance in psoriasis and HS, with and without concomitant IBD. Methods: Using quantitative polymerase chain reaction , we compared the F. prausnitzii and E. coli abundances in faecal samples from healthy controls [ n = 33] with samples from patients with psoriasis [ n = 29], IBD [ n = 31], and concomitant IBD and psoriasis [ n = 13]. Likewise, we analysed samples from patients with HS [ n = 17], and concomitant IBD and HS [ n = 17]. Results: Psoriasis patients harboured a significantly lower abundance of F. prausnitzii in their stool than healthy controls [ p < 0.001], which was similar to IBD patients. Together with the reduced F. prausnitzii levels, the psoriasis patients had a significantly higher abundance of E. coli [ p < 0.001]. No significant difference in F. prausnitzii or E. coli abundance was found in HS. It was apparent that patients with concomitant IBD and associated skin disorder had the greatest decrease of F. prausnitzii and increase of E. coli. Conclusions: The study demonstrates, for the first time, an IBD-like decrease of F. prausnitzii together with an increase of E.coli in psoriasis, supporting the presence of a gut-microbiome-skin axis in psoriasis and IBD.

The scientific interests in the colorectal cancer (CRC) associated microbiome have increased significantly in the past decade. Mechanistically, several members of the human microbiome and products thereof have been implicated as inductors of the pathogenic inflammation related to CRC. Conversely, the activities of the human intestinal microbial community influenced by specific diet might confer a protective effect against the CRC risks and progression. As the microbiome is both a key contributor and one of the tools to prevent CRC, the current review gives a summary of the CRC-associated microbiome and the dietary strategies relevant to CRC. As more evidences become available, new microbiome-based treatments and specific diets may emerge to reduce the CRC risk and improve CRC patients' quality of life.

In past decades, gut microbiome perturbations appeared to play a role in the development of several autoimmune diseases. Microbiome dysbiosis together with genetic, lifestyle and/or diet factors may composedly affect the etiology of autoimmune diseases. Studies about the role of the gut microbiome in psoriatic arthritis (PsA), a chronic autoimmune disease of the joint, are limited. We propose that intestinal dysbiosis-associated microbiota or gut microbiota-derived products might reach the joint by translocation to the circulation leading to T-cell activation and inflammation that underline PsA. Future studies on the intestinal microbiome associated with psoriatic arthritis can provide new targets for diagnostic and treatment strategies. Recognizing the PsA ‘early warning phases’ could offer new windows of opportunities regarding prevention.

Objective Although genome wide association studies have partly uncovered the genetic basis of Crohn's disease (CD), it remains a challenge to link genetic polymorphisms to functional intestinal phenotypes. Paneth cells are specialised antimicrobial epithelial cells localised to the small-intestinal crypt-base. Here, we investigate whether genomic variations in ATG16L1 affect Paneth cell function. Design Genomic variation of ATG16L1 (T300A, rs2241880) was determined in DNA from 78 patients with CD and 12 healthy controls. Paraffin-embedded ileal biopsies from patients with genotype AA (n=17), GA (n=38) and patients with the GG allele (n=23) were stained for GRP78, phospho-EIF2 alpha, lysozyme, cleaved-caspase 3, phosphohistone H3, phospho-I kappa B, p65, phospho-p38MAPK and PHLDA1. Microbial composition of biopsies was assessed by PCR. Disease phenotype was scored. Results In patients with quiescent disease but with an ATG16L1 risk allele, the endoplasmic reticulum (ER) stress markers GRP78 and pEIF2 alpha were highly expressed in Paneth cells. Other CD risk gene variations did not correlate with Paneth cell ER stress. Functionally, patients with ER-stressed Paneth cells showed no changes in intestinal epithelial cells proliferation or apoptosis, Paneth cell or stem cell numbers, p65, phospho-I kappa B and phospho-p38 staining. However, a significantly increased presence of adherent-invasive Escherichia coli was observed in biopsies from patients with ER-stressed Paneth cells. Phenotypically, patients with GRP78 positive Paneth cells have relatively less colonic disease over ileal disease (-21%, p=0.04), more fistulas (+21%, p=0.05) and an increased need for intestinal surgery (+38%, p=0.002). Conclusions The ATG16L1 T300A polymorphism defines a specific subtype of patients with CD, characterised by Paneth cell ER stress even during quiescent disease. Paneth cell ER stress correlates with bacterial persistence, and is thus likely to modulate antimicrobial functionality of this cell type in patients with CD.

Pregnancy has a beneficial influence on the course of certain autoimmune diseases such as inflammatory bowel disease (IBD). It was recently reported that during pregnancy the microbiome undergoes profound changes that are associated with host physiological and immunological adaptations. Here we propose that microbiome remodeling during pregnancy is an active response of the mother, possibly to alter immune system status, and to facilitate metabolic and immunological adaptations that are needed for a successful pregnancy. Furthermore, these changes in the microbiome may ensure the transfer of specific traits into the neonatal gut. As the underlying mechanisms are not well understood, elucidating how pregnancy-related changes in the microbiome influence IBD would be of obvious value for designing rational therapy.

Background Psoriasis and inflammatory bowel disease (IBD) are chronic inflammatory diseases sharing similar pathogenic pathways. Intestinal microbial changes such as a decrease of bakers’ yeast Saccharomyces cerevisiae have been reported in IBD, suggesting the presence of a gut-skin axis. Objective To investigate whether the S. cerevisiae abundance was altered in psoriasis patients versus healthy controls, and whether dimethylfumarate (DMF) interacted with this yeast. Methods Using qPCR, faecal samples were compared between psoriasis patients without DMF (n = 30), psoriasis patients with DMF (n = 28), and healthy controls (n = 32). Results Faecal S. cerevisiae abundance was decreased in psoriasis compared to healthy controls (p

Intestinal microbiota have emerged as an important factor in colorectal cancer (CRC) initiation and progression. The currently prominent view on bacterial tumorigenesis is that CRC initiation is triggered by local mucosal colonization with specific pathogens (drivers), and that subsequent changes in the peritumoral environment allow colonization by opportunistic (passenger) microbes, further facilitating disease progression. Screening for CRC 'driver-passenger' microorganisms might thus allow early CRC diagnosis or preventive intervention. Such efforts are now being revolutionized by the notion that CRC initiation and progression require organization of bacterial communities into higher-order structures termed biofilms. We explore here the concept that a polymicrobial biofilm promotes pro-carcinogenic activities that may partially underlie progression along the adenoma-CRC axis.

Prophylactic probiotic therapy has shown beneficial effects in an experimental rat model for acute pancreatitis on the health status of the animals. Mechanisms by which probiotic therapy interferes with severity of acute pancreatitis and associated sepsis, however, are poorly understood. The aims of this study were to identify the probiotic-induced changes in the gut microbiota and to correlate these changes to disease outcome. Duodenum and ileum samples were obtained from healthy and diseased rats subjected to pancreatitis for 7 days and prophylactically treated with either a multispecies probiotic mixture or a placebo. Intestinal microbiota was characterized by terminal-restriction fragment length polymorphism (T-RFLP) analyses of PCR-amplified 16S rRNA gene fragments. These analyses showed that during acute pancreatitis the host-specific ileal microbiota was replaced by an “acute pancreatitis-associated microbiota.” This replacement was not reversed by administration of the probiotic mixture. An increase, however, was observed in the relative abundance of a novel bacterial phylotype most closely related to Clostridium lituseburense and referred to as commensal rat ileum bacterium (CRIB). Specific primers targeting the CRIB 16S rRNA gene sequence were developed to detect this phylotype by quantitative PCR. An ileal abundance of CRIB 16S rRNA genes of more than 7.5% of the total bacterial 16S rRNA gene pool was correlated with reduced duodenal bacterial overgrowth, reduced bacterial translocation to remote organs, improved pancreas pathology, and reduced proinflammatory cytokine levels in plasma. Our current findings and future studies involving this uncharacterized bacterial phylotype will contribute to unraveling one of the potential mechanisms of probiotic therapy.

Summary Early-life exposure to appropriate microbial flora drives expansion and development of an efficient immune system. Aberrant development results in increased likelihood of allergic disease or increased susceptibility to infection. Thus, factors affecting microbial colonization may also affect the direction of immune responses in later life. There is a need for a manipulable animal model of environmental influences on the development of microbiota and the immune system during early life. We assessed the effects of rearing under low- (farm, sow) and high-hygiene (isolator, milk formula) conditions on intestinal microbiota and immune development in neonatal piglets, because they can be removed from the mother in the first 24 h for rearing under controlled conditions and, due to placental structure, neither antibody nor antigen is transferred in utero. Microbiota in both groups was similar between 2 and 5 days. However, by 12–28 days, piglets reared on the mother had more diverse flora than siblings reared in isolators. Dendritic cells accumulated in the intestinal mucosa in both groups, but more rapidly in isolator piglets. Importantly, the minority of 2–5-day-old farm piglets whose microbiota resembled that of an older (12–28-day-old) pig also accumulated dendritic cells earlier than the other farm-reared piglets. Consistent with dendritic cell control of T cell function, the effects on T cells occurred at later time-points, and mucosal T cells from high-hygiene, isolator pigs made less interleukin (IL)-4 while systemic T cells made more IL-2. Neonatal piglets may be a valuable model for studies of the effects of interaction between microbiota and immune development on allergy.

The microbial community in the guts of mammals is often seen as an important potential target in therapeutic and preventive interventions. The aim of the present study was to determine whether enterotoxigenic Escherichia coli (ETEC) F4 infection in young animals might be counteracted by a probiotic treatment with Lactobacillus sobrius DSM 16698. The experiment was conducted in three randomized consecutive replications, each consisting of 16 piglets, and including a control group and an L. sobrius fed group, both experimentally challenged with ETEC. During the entire trial, the animals' health status, body weight, and microbial parameters were monitored periodically. Probiotic supplementation containing L. sobrius significantly reduced the levels of ETEC in the ileum when fed directly to piglets after weaning. In contrast, the number of days when the piglets had an increased faecal water content was significantly higher in the probiotic group. Nevertheless, an improved daily weight gain was also observed in the animals that received probiotic L. sobrius relative to the control fed group. The data indicate that L. sobrius may be effective in the reduction of the E. coli F4 colonization and may improve the weight gain of infected piglets.

Background Mortality during severe acute pancreatitis generally occurs early or late. Early mortality occurs as a consequence of fulminant systemic inflammation, whereas late mortality can be attributed to secondary infection of necrotic pancreatic tissue. Aims of this study were to correlate plasma levels of various cytokines and chemokines to outcome, and secondly, to assess the effect of probiotic modulation of intestinal flora on systemic cytokine levels. Materials and methods Pancreatitis was induced in rats by intraductal infusion of bile salt followed by i.v. cerulein hyperstimulation. Rats were administered a mixture of 6 probiotic strains ( Ecologic ® 641) or placebo for five days prior to and seven days after induction of pancreatitis. Plasma cytokine levels were determined before, and 6 hours, 24 hours and 7 days after induction of pancreatitis. Total bacterial counts were performed by cultivation as well as by cultivation-independent rt-PCR in pancreatic necrosis at day 7.

Lactobacillus sobrius sp. nov., which was recently isolated from the intestine of weaning piglets, has potential probiotic properties. To follow the fate of L. sobrius strain 001 T in dietary interventions, a novel and strain-specific quantitative detection procedure was developed. This procedure was based on the isolation of specific genomic fragments from the type strain by representational difference analysis and their detection by real-time PCR. The described strain-specific quantification approach may be used in studies aimed at tracking bacterial strains added to specific environments.

Recently, it has become apparent that GIT fermentation is not only of interest for ruminant animals, but also for monogastrics. While it is now widely accepted that the fermentation process and its resultant end-products can have important influences on animal health, little is known about the microbiological and immunological processes involved. In terms of animal health, most interest at the moment is focussed on those moments in animals' lives when they are faced with sudden changes resulting in stress. The period of weaning in piglets is a typical example of this. The most easily accomplished and appropriate way to influence GIT fermentation processes is that of dietary intervention. This is reflected by the widespread interest in so-called pre- and pro-biotics. Given the complexities of the interactions occurring in the animal itself, it is hardly surprising that in vitro techniques are being widely used: firstly to examine potential substrates for their fermentability and possible inclusion in diets, and secondly, to assess changes in the microbial populations in response to these substrates. This paper will review the techniques currently in use for these two aspects of monogastric fermentation, and provide examples of their use.

In vitro fermentation of sugar beet pulp (SBP) was carried out to determine which bacterial species would be enriched by use of this carbohydrate source. Faeces from four weaning piglets as a source of inoculum was also compared. The microbial diversity of the prominent bacteria before and after this in vitro fermentation was analysed using denaturing gradient gel electrophoresis (DGGE) of PCR amplicons of 16S rDNA. Before fermentation, the DGGE profiles showed differences between cultures inoculated with faeces from different piglets, though some bands were common to all piglets. After fermentation of SBP, three dominant bands appeared, of which two bands appeared in all samples and one for both replicates of one piglet. Sequences of the corresponding 16S rDNA of two bands showed 92% similarity to Eubacterium eligens and 96% similarity to Lachnospira pectinoschiza, and that of the third band 95% to L. pectinoschiza. (C) 2003 Elsevier Ltd. All rights reserved.

The effect of fermentable carbohydrates (sugar beet pulp and fructooligosaccharides) on the faecal bacterial communities of weaning piglets was analysed using 16S rDNA-based approaches. Amplicons of the V6-V8 variable regions of bacterial 16S rDNA were analysed by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. Differences in piglet faecal bacterial community structure were determined based on the Dice coefficients for pairwise comparison of the DGGE fingerprints and revealed significant changes in the faecal microbiota immediately after weaning. Piglets fed with fermentable carbohydrates showed a higher bacterial diversity and a more rapid stabilisation of the bacterial community compared with that of the animals fed with the control diet. Thirteen dominant DGGE bands were matched with sequences that showed 91-97% similarity to those derived from the Clostridium coccoides group and the Clostridium leptum subgroup. Amplicons related to Ruminococcus-like species were found in all DGGE fingerprints derived from pigs on the diet containing sugar beet pulp and fructooligosaccharides, but not in pigs on the control diet. These results indicate that these bacteria may play a role in the utilisation of dietary fibres.

In this chapter the term probiotics is used in a broad sense to include the use of various different microbes in veterinary strategies aimed at manipulation or replacement of intestinal microbes. The reduction in the incidence of diarrhea by probiotics has frequently been studied because this problem is of the utmost importance for farm animals. Neonatal farmhouse animals including piglets often have an insufficiency of stomach acid, which is the first line of defense against bacterial invasion. Microbes of all types, including bacteria, viruses, archaea, fungi, and protozoans, colonize and persist in the intestinal tract of farm animals. Genomic analysis of differential gene expression in microbes and higher organisms has been greatly facilitated by the development of DNA microarrays. This technology has been exploited to monitor global intestinal transcriptional responses to the colonization of germ free mice with Bacteroides thetaiotaomicron, a prominent member of the normal murine and human intestinal microbiota. This study showed that this commensal was able to modulate expression of host genes participating in diverse and fundamental physiological functions, including nutrient absorption, mucosal barrier fortification, xenobiotic metabolism, angiogenesis, and postnatal intestinal maturation. Recent in vitro studies have suggested that probiotics can have protective effects on cytokine-induced apoptosis or permeability changes, attenuate epithelial inflammatory responses, and reduce invasion or adherence of pathogens.

Psoriatic arthritis is a chronic inflammatory joint disease, seen in combination with the chronic inflammatory skin disease psoriasis and belonging to the family of spondylarthritides (SpA). A link is recognized between psoriatic arthritis and inflammatory bowel disease (IBD). Environmental factors seem to induce inflammatory disease in individuals with underlying genetic susceptibility. The microbiome is a subject of increasing interest in the etiology of these inflammatory immune-mediated diseases. The intestinal microbiome is able to affect extra-intestinal distant sites, including the joints, through immunomodulation. At this point, evidence regarding a relationship between the microbiome and psoriatic arthritis is scarce. However, we hypothesize that common immune-mediated inflammatory pathways seen in the "skin-joint-gut axis" in psoriatic arthritis are induced or at least mediated by the microbiome. Th17 has a crucial function in this mechanism. Further establishment of this connection may lead to novel therapeutic approaches for psoriatic arthritis.

Colorectal cancer may develop slowly over years from precursor lesions, and thus screening combined with early diagnosis is the key to disease prevention. Recent studies have elucidated specific traits in the gut microbiome associated with colorectal cancer and suggested that the microbiome may be useful in screening for colorectal cancer purposes but failed to provide protocols that can be applied in a practical situation. A recent study by Zackular and colleagues, presented on page 1112, provides an important way forward here in showing that specific analysis of multiple aspects of the microbiome composition in toto provides reliable detection of both precancerous and cancerous lesions. This important achievement when combined with other noninvasive techniques promises to provide highly effective tools for early colorectal cancer diagnosis and its prevention. Cancer Prev Res; 7(11); 1108–11. ©2014 AACR.

The change of the bacteria from colonizers to pathogens is accompanied by a drastic change in expression profiles. These changes may be due to environmental signals or to mutational changes. We therefore compared the whole genome sequences of four sets of S. aureus isolates. Three sets were from the same patients. The isolates of each pair (S1800/S1805, S2396/S2395, S2398/S2397, an isolate from colonization and an isolate from infection, respectively) were obtained within

The evidence for a strong correlation between the gut microbiota and colorectal carcinogenesis is quickly gathering pace. This correlation raises important questions, such as whether analysis of the microbiota can be used for screening purposes, and whether targeted intervention can influence the risk of development and progression of neoplasia. The recovery of several pathobionts-such as members of the different bacterial phyla Proteobacteria, Bacteroidetes and Fusobacteria-from the tumour microenvironment of patients with colorectal cancer (CRC) now provides a link between specific microbial colonization and cancer. However, other intestinal bacteria belonging to another major intestinal phylum, Firmicutes, might be effective in the treatment of pathogenic inflammation related to CRC. Future approaches based on the analysis of the gut microbiota of patients with CRC combined with large human cohort studies might open up new possibilities for further prophylactic, screening and treatment strategies.

Dendritic cells (DCs) are antigen-presenting cells that play an essential role in mucosal tolerance. They regularly encounter beneficial intestinal bacteria, but the nature of these cellular contacts and the immune responses elicited by the bacteria are not entirely elucidated. Here, we examined the interactions of Lactobacillus acidophilus NCFM and its cell surface compounds with DCs. L. acidophilus NCFM attached to DCs and induced a concentration-dependent production of IL-10, and low IL-12p70. We further demonstrated that the bacterium binds to DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a DC- specific receptor. To identify the DC-SIGN ligand present on the bacterium, we took advantage of a generated array of L. acidophilus NCFM mutants. A knockout mutant of L. acidophilus NCFM lacking the surface (S) layer A protein (SlpA) was significantly reduced in binding to DC-SIGN. This mutant incurred a chromosomal inversion leading to dominant expression of a second S layer protein, SlpB. In the SlpB-dominant strain, the nature of the interaction of this bacterium with DCs changed dramatically. Higher concentrations of proinflammatory cytokines such as IL-12p70, TNFα, and IL-1β were produced by DCs interacting with the SlpB-dominant strain compared with the parent NCFM strain. Unlike the SlpA-knockout mutant, T cells primed with L. acidophilus NCFM stimulated DCs produced more IL-4. The SlpA–DC-SIGN interaction was further confirmed as purified SlpA protein ligated directly to the DC-SIGN. In conclusion, the major S layer protein, SlpA, of L. acidophilus NCFM is the first probiotic bacterial DC-SIGN ligand identified that is functionally involved in the modulation of DCs and T cells functions.

Lactobacilli have a potential to overcome intestinal disorders; however, the exact mode of action is still largely unknown. In this study, we have used the intestinal porcine intestinal IPEC-1 epithelial cells as a model to investigate a possible protective activity of a new Lactobacillus species, the L. sobrius DSM 16698(T), against intestinal injury induced by enterotoxigenic Escherichia coli (ETEC) K88 infection and the underlying mechanisms. Treatment of infected cells with L. sobrius strongly reduced the pathogen adhesion. L. sobrius was also able to prevent the ETEC-induced membrane damage by inhibiting delocalization of zonula occludens (ZO)-1, reduction of occludin amount, rearrangement of F-actin, and dephosphorylation of occludin caused by ETEC. RT-PCR and ELISA experiments showed that L. sobrius counteracted the ETEC-induced increase of IL-8 and upregulated the IL-10 expression. The involvement of IL-8 in the deleterious effects of ETEC was proven by neutralization of IL-8 with a specific antibody. A crucial role of IL-10 was indicated by blockage of IL-10 production with neutralizing anti-IL-10 antibody that fully abrogated the L. sobrius protection. L. sobrius was also able to inhibit the internalization of ETEC, which was likely favored by the leaking barrier. The protective effects were not found with L. amylovorus DSM 20531(T) treatment, a strain derived from cattle waste but phylogenetically closely related to L. sobrius. Together, the data indicate that L. sobrius exerts protection against the harmful effects of ETEC by different mechanisms, including pathogen adhesion inhibition and maintenance of membrane barrier integrity through IL-10 regulation.

Infection of pancreatic necrosis by gut bacteria is a major cause of morbidity and mortality in patients with severe acute pancreatitis. Use of prophylactic antibiotics remains controversial. The aim of this experiment was assess if modification of intestinal flora with specifically designed multispecies probiotics reduces bacterial translocation or improves outcome in a rat model of acute pancreatitis. METHODS: Male Sprague-Dawley rats were allocated into 3 groups: (1) controls (sham-operated, no treatment), (2) pancreatitis and placebo, and (3) pancreatitis and probiotics. Acute pancreatitis was induced by intraductal glycodeoxycholate and intravenous cerulein infusion. Daily probiotics or placebo was administered intragastrically from 5 days prior until 7 days after induction of pancreatitis. Tissue and fluid samples were collected for microbiologic and quantitative real-time PCR analysis of bacterial translocation. RESULTS: Probiotics reduced duodenal bacterial overgrowth of potential pathogens (Log(10) colony-forming units [CFU]/g 5.0 +/- 0.7 [placebo] vs 3.5 +/- 0.3 CFU/g [probiotics], P

The in vitro cumulative gas production technique can be used to assess microbial activity of a complex community, in relation to fermentation of a particular energy source. Therefore, in combination with an in vivo study to examine the effects of two different diets for weaning piglets, microbial activities of faeces were compared from animals on the two different diets. The two diets were: CHO diet [containing added fermentable carbohydrates, including sugarbeet pulp (SBP) and wheat starch (WST)], and control diet without any added fermentable carbohydrates. Neither diet contained antibiotics or extra added copper. Twenty-four piglets were selected from 12 litters (two per litter), weaned at 4 weeks of age (neither creep feeding nor any antibiotic treatment before and during the study), and introduced to one of the two diets. After 9 days on the diet, faecal samples were collected from selected animals, and tested for their activity in terms of gas production kinetics, and end-products such as volatile fatty acids, ammonia and dry matter disappearance of the two test substrates SBP and WST. The bacterial diversity was also analysed before and after in vitro fermentation using denaturing gradient gel electrophoresis analysis of amplified 16S rRNA genes. There were differences both in kinetics and end-products of the substrates. More interestingly, significant differences were detected between inocula, although mainly in terms of fermentation kinetics of the two substrates. With the CHO inoculum, SBP was fermented faster than with the control, while this effect was reversed for WST. Significantly higher diversity, as measured by DGGE fingerprint analysis, was detected in the microbial community enrichment on SBP as compared with WST at the end of fermentation. The difference between the kinetics of SBP compared with WST fermentation by faecal microbiota from the CHO diet fed piglets suggests better adaptation to SBP fermentation than to WST fermentation. The WST fermentation was more unexpected, given that a significant amount of starch is known to be fermentable by the small intestinal microbiota. It was concluded that the microbial community composition and activity in the GIT may be changed in response to diet, and that this change can be detected in vitro. Copyright © 2005 Society of Chemical Industry

Using 16S rRNA gene-based approaches, we analyzed the responses of ileal and colonic bacterial communities of weaning piglets to dietary addition of four fermentable carbohydrates (inulin, lactulose, wheat starch, and sugar beet pulp). An enriched diet and a control diet lacking these fermentable carbohydrates were fed to piglets for 4 days ( n = 48), and 10 days ( n = 48), and the lumen-associated microbiota were compared using denaturing gradient gel electrophoresis (DGGE) analysis of amplified 16S rRNA genes. Bacterial diversities in the ileal and colonic samples were measured by assessing the number of DGGE bands and the Shannon index of diversity. A higher number of DGGE bands in the colon (24.2 ± 5.5) than in the ileum (9.7 ± 4.2) was observed in all samples. In addition, significantly higher diversity, as measured by DGGE fingerprint analysis, was detected in the colonic microbial community of weaning piglets fed the fermentable-carbohydrate-enriched diet for 10 days than in the control. Selected samples from the ileal and colonic lumens were also investigated using fluorescent in situ hybridization (FISH) and cloning and sequencing of the 16S rRNA gene. This revealed a prevalence of Lactobacillus reuteri in the ileum and Lactobacillus amylovorus -like populations in the ileum and the colon in the piglets fed with fermentable carbohydrates. Newly developed oligonucleotide probes targeting these phylotypes allowed their rapid detection and quantification in the ileum and colon by FISH. The results indicate that addition of fermentable carbohydrates supports the growth of specific lactobacilli in the ilea and colons of weaning piglets.

At the time of weaning, major quantitative and qualitative changes occur in the composition of the intestinal microbiota of piglets, influenced by diet, environmental factors, and the host. Within a short period of time, the intestinal microbiota must ultimately develop from a simple, unstable community into a complex and stable one. Here we present data on the development of the intestinal microbiota based on 16S rRNA gene sequence diversity. In addition to a PCR-based analysis of the 16S rRNA gene by cloning and denaturing gradient gel electrophoresis (DGGE), data on fluorescent in situ hybridisation ( FISH) are presented to quantify the total bacterial communities, major Lactobacillus populations and specific Lactobacillus species. The results reported here indicate that the addition of non-digestible, fermentable carbohydrates (= prebiotics) leads to an enrichment of lactobacilli in the small intestine, and increased stability and diversity of the bacterial community in the colon. The data support the hypothesis that changes of the diet can modulate the composition of the microbiota in the intestine. These findings may have potentially major implications for the development of dietary strategies aiming to improve animal health during the weaning process.

Gram-negative sepsis is a potentially fatal clinical syndrome characterized by a proinflammatory response (tumor necrosis factor-alpha) to bacterial (endo)toxins and gut barrier function loss. Recently, we found that high-fat enteral nutrition protects against late bacterial translocation in a model of hemorrhagic shock in rats. However, the basis for this protection is unknown. We hypothesized that the observed protection is the result of an early inhibition of endotoxin and the subsequent inflammatory response resulting in a preserved gut barrier function. Sprague-Dawley rats were divided into a group that was starved overnight (HS-S), fed with a low-fat enteral diet (HS-LF) or fed wih a high-fat enteral diet (HS-HF), and subsequently subjected to a nonlethal hemorrhagic shock. Ninety minutes after hemorrhage, arterial endotoxin significantly decreased in HS-HF rats (4.0 +/- 0.6 pg/mL) compared with HS-LF rats (10.7 +/- 0.9 pg/mL, P = 0.002) and HS-S rats (15.2 +/- 2.2 pg/mL P = 0.001). Interestingly, arterial tumor necrosis factor-alpha was also decreased in HS-HF rats (17.9 +/- 10.4 pg/mL) compared with HS-LF (83.5 +/- 16.7 pg/mL, P < 0.01) and HS-S rats (180.9 +/- 67.9 pg/mL, P < 0.02). Loss of tight junction structure (ZO-1) observed in ileum and colon of control hemorrhagic shock rats was prevented in HS-HF rats. In parallel, intestinal barrier function was preserved in HS-HF rats, evidenced by a reduced permeability to horseradish peroxidase (P < 0.05), less bacterial invasion, and a 10-fold reduction of bacterial translocation early after hemorrhagic shock. This report describes a new strategy to nutritionally prevent endotoxemia, the subsequent inflammatory response and gut barrier failure following hemorrhagic shock. High-fat enteral nutrition requires further evaluation as an intervention to prevent a potentially fatal systemic inflammatory response in patients at risk for sepsis.