Modulation of Gut Microbiota Composition by Serotonin Signaling Influences Intestinal Immune Response and Susceptibility to Colitis.

BACKGROUND & AIMS: Serotonin (5-hydroxytryptamine [5-HT]) is synthesized mainly within enterochromaffin (EC) cells in the gut, and tryptophan hydroxylase 1 (Tph1) is the rate-limiting enzyme for 5-HT synthesis in EC cells. Accumulating evidence suggests the importance of gut microbiota in intestinal inflammation. Considering the close proximity of EC cells and the microbes, we investigated the influence of gut-derived 5-HT on the microbiota and the susceptibility to colitis. METHODS: Gut microbiota of Tph1-/- and Tph1+/- mice were investigated by deep sequencing. Direct influence of 5-HT on bacteria was assessed by using in vitro system of isolated commensals. The indirect influence of 5-HT on microbiota was assessed by measuring antimicrobial peptides, specifically ß-defensins, in the colon of mice and HT-29 colonic epithelial cells. The impact of gut microbiota on the development of dextran sulfate sodium-induced colitis was assessed by transferring gut microbiota from Tph1-/- mice to Tph1+/- littermates and vice versa, as well as in germ-free mice. RESULTS: A significant difference in microbial composition between Tph1-/- and Tph1+/- littermates was observed. 5-HT directly stimulated and inhibited the growth of commensal bacteria in vitro, exhibiting a concentration-dependent and species-specific effect. 5-HT also inhibited ß-defensin production by HT-29 cells. Microbial transfer from Tph1-/- to Tph1+/- littermates and vice versa altered colitis severity, with microbiota from Tph1-/- mice mediating the protective effects. Furthermore, germ-free mice colonized with microbiota from Tph1-/- mice exhibited less severe dextran sulfate sodium-induced colitis. CONCLUSIONS: These findings demonstrate a novel role of gut-derived 5-HT in shaping gut microbiota composition in relation to susceptibility to colitis, identifying 5-HT-microbiota axis as a potential new therapeutic target in intestinal inflammatory disorders.

Targeting macrophage scavenger receptor 1 promotes insulin resistance in obese male mice.

Immune components can bridge inflammatory triggers to metabolic dysfunction. Scavenger receptors sense lipoproteins, but it is not clear how different scavenger receptors alter carbohydrate metabolism during obesity. Macrophage scavenger receptor 1 (MSR1) and macrophage receptor with collagenous structure (MARCO) are scavenger receptors that have been implicated in lipoprotein metabolism and cardiovascular disease. We assessed glucose control, tissue-specific insulin sensitivity, and inflammation in Msr1- and Marco-deficient mice fed with obesogenic diets. Compared to wild-type (WT) mice, Msr1-/- mice had worse blood glucose control that was only revealed after diet-induced obesity, not in lean mice. Obese Msr1-/- mice had worse insulin-stimulated glucose uptake in the adipose tissue, which occurred in the absence of overt differences in adipose inflammation compared to obese WT mice. Msr1 deletion worsened dysglycemia independently from bacterial cell wall insulin sensitizers, such as muramyl dipeptide. MARCO was dispensable for glycemic control in obese mice. Oral administration of the polysaccharide fucoidan worsened glucose control in obese WT mice, but fucoidan had no effect on glycemia in obese Msr1-/- mice. Therefore, MSR1 is a scavenger receptor responsible for changes in glucose control in response to the environmental ligand fucoidan. Given the interest in dietary supplements and natural products reducing inflammation or insulin resistance in metabolic disease during obesity, our results highlight the importance of understanding which ligand-receptor relationships promote versus those that protect against metabolic disease factors. Our results show that ligand or gene targeting of MSR1 exacerbates insulin resistance in obese mice.

Long term but not short term exposure to obesity related microbiota promotes host insulin resistance.

The intestinal microbiota and insulin sensitivity are rapidly altered after ingestion of obesogenic diets. We find that changes in the composition of the fecal microbiota precede changes in glucose tolerance when mice are fed obesogenic, low fiber, high fat diets (HFDs). Antibiotics alter glycemia during the first week of certain HFDs, but antibiotics show a more robust improvement in glycemic control in mice with protracted obesity caused by long-term feeding of multiple HFDs. Microbiota transmissible dysglycemia and glucose intolerance only occur when germ-free mice are exposed to obesity-related microbes for more than 45 days. We find that sufficient host exposure time to microbiota derived from HFD-fed mice allows microbial factors to contribute to insulin resistance, independently from increased adiposity in mice. Our results are consistent with intestinal microbiota contributing to chronic insulin resistance and dysglycemia during prolonged obesity, despite rapid diet-induced changes in the taxonomic composition of the fecal microbiota.

Muramyl Dipeptide-Based Postbiotics Mitigate Obesity-Induced Insulin Resistance via IRF4.

Intestinal dysbiosis contributes to obesity and insulin resistance, but intervening with antibiotics, prebiotics, or probiotics can be limited by specificity or sustained changes in microbial composition. Postbiotics include bacterial components such as lipopolysaccharides, which have been shown to promote insulin resistance during metabolic endotoxemia. We found that bacterial cell wall-derived muramyl dipeptide (MDP) is an insulin-sensitizing postbiotic that requires NOD2. Injecting MDP lowered adipose inflammation and reduced glucose intolerance in obese mice without causing weight loss or altering the composition of the microbiome. MDP reduced hepatic insulin resistance during obesity and low-level endotoxemia. NOD1-activating muropeptides worsened glucose tolerance. IRF4 distinguished opposing glycemic responses to different types of peptidoglycan and was required for MDP/NOD2-induced insulin sensitization and lower metabolic tissue inflammation during obesity and endotoxemia. IRF4 was dispensable for exacerbated glucose intolerance via NOD1. Mifamurtide, an MDP-based drug with orphan drug status, was an insulin sensitizer at clinically relevant doses in obese mice.

Resveratrol improves exercise performance and skeletal muscle oxidative capacity in heart failure.

We investigated whether treatment of mice with established pressure overload-induced heart failure (HF) with the naturally occurring polyphenol resveratrol could improve functional symptoms of clinical HF such as fatigue and exercise intolerance. C57Bl/6N mice were subjected to either sham or transverse aortic constriction surgery to induce HF. Three weeks postsurgery, a cohort of mice with established HF (%ejection fraction <45) was administered resveratrol (~450 mg·kg-1·day-1) or vehicle for 2 wk. Although the percent ejection fraction was similar between both groups of HF mice, those mice treated with resveratrol had increased total physical activity levels and exercise capacity. Resveratrol treatment was associated with altered gut microbiota composition, increased skeletal muscle insulin sensitivity, a switch toward greater whole body glucose utilization, and increased basal metabolic rates. Although muscle mass and strength were not different between groups, mice with HF had significant declines in basal and ADP-stimulated O2 consumption in isolated skeletal muscle fibers compared with sham mice, which was completely normalized by resveratrol treatment. Overall, resveratrol treatment of mice with established HF enhances exercise performance, which is associated with alterations in whole body and skeletal muscle energy metabolism. Thus, our preclinical data suggest that resveratrol supplementation may effectively improve fatigue and exercise intolerance in HF patients.NEW & NOTEWORTHY Resveratrol treatment of mice with heart failure leads to enhanced exercise performance that is associated with altered gut microbiota composition, increased whole body glucose utilization, and enhanced skeletal muscle metabolism and function. Together, these preclinical data suggest that resveratrol supplementation may effectively improve fatigue and exercise intolerance in heart failure via these mechanisms.

Improved Glucose Homeostasis in Obese Mice Treated With Resveratrol Is Associated With Alterations in the Gut Microbiome.

Oral administration of resveratrol is able to improve glucose homeostasis in obese individuals. Herein we show that resveratrol ingestion produces taxonomic and predicted functional changes in the gut microbiome of obese mice. In particular, changes in the gut microbiome were characterized by a decreased relative abundance of Turicibacteraceae, Moryella, Lachnospiraceae, and Akkermansia and an increased relative abundance of Bacteroides and Parabacteroides Moreover, fecal transplantation from healthy resveratrol-fed donor mice is sufficient to improve glucose homeostasis in obese mice, suggesting that the resveratrol-mediated changes in the gut microbiome may play an important role in the mechanism of action of resveratrol.

High-intensity exercise training increases the diversity and metabolic capacity of the mouse distal gut microbiota during diet-induced obesity.

Diet and exercise underpin the risk of obesity-related metabolic disease. Diet alters the gut microbiota, which contributes to aspects of metabolic disease during obesity. Repeated exercise provides metabolic benefits during obesity. We assessed whether exercise could oppose changes in the taxonomic and predicted metagenomic characteristics of the gut microbiota during diet-induced obesity. We hypothesized that high-intensity interval training (HIIT) would counteract high-fat diet (HFD)-induced changes in the microbiota without altering obesity in mice. Compared with chow-fed mice, an obesity-causing HFD decreased the Bacteroidetes-to-Firmicutes ratio and decreased the genetic capacity in the fecal microbiota for metabolic pathways such as the tricarboxylic acid (TCA) cycle. After HFD-induced obesity was established, a subset of mice were HIIT for 6 wk, which increased host aerobic capacity but did not alter body or adipose tissue mass. The effects of exercise training on the microbiota were gut segment dependent and more extensive in the distal gut. HIIT increased the alpha diversity and Bacteroidetes/Firmicutes ratio of the distal gut and fecal microbiota during diet-induced obesity. Exercise training increased the predicted genetic capacity related to the TCA cycle among other aspects of metabolism. Strikingly, the same microbial metabolism indexes that were increased by exercise were all decreased in HFD-fed vs. chow diet-fed mice. Therefore, exercise training directly opposed some of the obesity-related changes in gut microbiota, including lower metagenomic indexes of metabolism. Some host and microbial pathways appeared similarly affected by exercise. These exercise- and diet-induced microbiota interactions can be captured in feces.

Different Th17 immunity in gut, liver, and adipose tissues during obesity: the role of diet, genetics, and microbes.

Microbes modify immunometabolism responses linking obesity and type 2 diabetes. Immunity helps maintain a host-microbe symbiosis, but inflammation can promote insulin resistance in tissues that control blood glucose. We were interested in compartmentalization of immune responses during obesity and show here that feeding mice an obesity-causing high-fat diet (HFD) decreased a marker of neutrophil activation and cytokines related to Th17 responses in the gut. A HFD decreased IL-17 and IL-21/22 in the ileum and colon, respectively. A HFD increased IL-17, IL-21/22 and other related Th17 responses in the liver. At the whole tissue level, there is divergence in gut and metabolic tissue Th17 cytokines during diet-induced obesity. Deletion of the bacterial peptidoglycan sensor NOD2 had relatively minor effects on these immune responses. We propose a model where diet-induced obesity promotes a permissive gut immune environment and sets the stage for host genetics to contribute to dysbiosis-driven metabolic tissue inflammation.

New Role of Nod Proteins in Regulation of Intestinal Goblet Cell Response in the Context of Innate Host Defense in an Enteric Parasite Infection.

Mucins secreted by intestinal goblet cells are considered an important component of innate defense in a number of enteric infections, including many parasitic infections, but also likely provide protection against the gut microbiota. Nod proteins are intracellular receptors that play key roles in innate immune response and inflammation. Here, we investigated the role of Nod proteins in regulation of intestinal goblet cell response in naive mice and mice infected with the enteric parasite Trichuris muris. We observed significantly fewer periodic acid-Schiff (PAS)-stained intestinal goblet cells and less mucin (Muc2) in Nod1 and Nod2 double-knockout (Nod DKO) mice after T. muris infection than in wild-type (WT) mice. Expulsion of parasites from the intestine was significantly delayed in Nod DKO mice. Treatment of naive WT mice with Nod1 and Nod2 agonists simultaneously increased numbers of PAS-stained goblet cells and Muc2-expressing cells, whereas treatment with Nod1 or Nod2 separately had no significant effect. Stimulation of mucin-secreting LS174T cells with Nod1 and Nod2 agonists upregulated core 3 ß1,3-N-acetylglucosaminyltransferase (C3GnT; an important enzyme in mucin synthesis) and MUC2. We also observed lower numbers of PAS-stained goblet cells and less Muc2 in germfree mice. Treatment with Nod1 and Nod2 agonists enhanced the production of PAS-stained goblet cells and Muc2 in germfree mice. These data provide novel information on the role of Nod proteins in goblet cell response and Muc2 production in relation to intestinal innate defense.

Defective NOD2 peptidoglycan sensing promotes diet-induced inflammation, dysbiosis, and insulin resistance.

Pattern recognition receptors link metabolite and bacteria-derived inflammation to insulin resistance during obesity. We demonstrate that NOD2 detection of bacterial cell wall peptidoglycan (PGN) regulates metabolic inflammation and insulin sensitivity. An obesity-promoting high-fat diet (HFD) increased NOD2 in hepatocytes and adipocytes, and NOD2(-/-) mice have increased adipose tissue and liver inflammation and exacerbated insulin resistance during a HFD. This effect is independent of altered adiposity or NOD2 in hematopoietic-derived immune cells. Instead, increased metabolic inflammation and insulin resistance in NOD2(-/-) mice is associated with increased commensal bacterial translocation from the gut into adipose tissue and liver. An intact PGN-NOD2 sensing system regulated gut mucosal bacterial colonization and a metabolic tissue dysbiosis that is a potential trigger for increased metabolic inflammation and insulin resistance. Gut dysbiosis in HFD-fed NOD2(-/-) mice is an independent and transmissible factor that contributes to metabolic inflammation and insulin resistance when transferred to WT, germ-free mice. These findings warrant scrutiny of bacterial component detection, dysbiosis, and protective immune responses in the links between inflammatory gut and metabolic diseases, including diabetes.

Interleukin-15 modulates adipose tissue by altering mitochondrial mass and activity.

Interleukin-15 (IL-15) is an immunomodulatory cytokine that affects body mass regulation independent of lymphocytes; however, the underlying mechanism(s) involved remains unknown. In an effort to investigate these mechanisms, we performed metabolic cage studies, assessed intestinal bacterial diversity and macronutrient absorption, and examined adipose mitochondrial activity in cultured adipocytes and in lean IL-15 transgenic (IL-15tg), overweight IL-15 deficient (IL-15-/-), and control C57Bl/6 (B6) mice. Here we show that differences in body weight are not the result of differential activity level, food intake, or respiratory exchange ratio. Although intestinal microbiota differences between obese and lean individuals are known to impact macronutrient absorption, differing gut bacteria profiles in these murine strains does not translate to differences in body weight in colonized germ free animals and macronutrient absorption. Due to its contribution to body weight variation, we examined mitochondrial factors and found that IL-15 treatment in cultured adipocytes resulted in increased mitochondrial membrane potential and decreased lipid deposition. Lastly, IL-15tg mice have significantly elevated mitochondrial activity and mass in adipose tissue compared to B6 and IL-15-/- mice. Altogether, these results suggest that IL-15 is involved in adipose tissue regulation and linked to altered mitochondrial function.

Fluvastatin causes NLRP3 inflammasome-mediated adipose insulin resistance.

Statins reduce lipid levels and are widely prescribed. Statins have been associated with an increased incidence of type 2 diabetes, but the mechanisms are unclear. Activation of the NOD-like receptor family, pyrin domain containing 3 (NLRP3)/caspase-1 inflammasome, promotes insulin resistance, a precursor of type 2 diabetes. We showed that four different statins increased interleukin-1ß (IL-1ß) secretion from macrophages, which is characteristic of NLRP3 inflammasome activation. This effect was dose dependent, absent in NLRP3(-/-) mice, and prevented by caspase-1 inhibition or the diabetes drug glyburide. Long-term fluvastatin treatment of obese mice impaired insulin-stimulated glucose uptake in adipose tissue. Fluvastatin-induced activation of the NLRP3/caspase-1 pathway was required for the development of insulin resistance in adipose tissue explants, an effect also prevented by glyburide. Fluvastatin impaired insulin signaling in lipopolysaccharide-primed 3T3-L1 adipocytes, an effect associated with increased caspase-1 activity, but not IL-1ß secretion. Our results define an NLRP3/caspase-1-mediated mechanism of statin-induced insulin resistance in adipose tissue and adipocytes, which may be a contributing factor to statin-induced development of type 2 diabetes. These results warrant scrutiny of insulin sensitivity during statin use and suggest that combination therapies with glyburide, or other inhibitors of the NLRP3 inflammasome, may be effective in preventing the adverse effects of statins.

Interleukin 13 and serotonin: linking the immune and endocrine systems in murine models of intestinal inflammation.

OBJECTIVE: Infiltration of activated immune cells and increased cytokine production define the immunophenotype of gastrointestinal (GI) inflammation. In addition, intestinal inflammation is accompanied by alteration in the numbers of serotonin (5-hydroxytryptamine; 5-HT) synthesizing enterochromaffin (EC) cells and in 5-HT amount. It has been established that EC cells express interleukin (IL)-13 receptor, additionally IL-13 has been implicated in the pathogenesis of ulcerative colitis. In this study, we investigated the role of IL-13 mediated 5-HT signaling in pathogenesis of colitis. METHODOLOGY: Colitis was induced in IL-13 deficient (IL-13-/-) and wild-type (WT) mice with dextran sulfate sodium (DSS) and dinitrobenzene sulfonic acid (DNBS), as well as in IL-13-/- mice given recombinant mouse IL-13 (rmIL-13) and 5-hydroxytryptamine (5-HTP), the direct precursor of 5-HT. PRINCIPAL FINDINGS AND CONCLUSION: Elevated colonic IL-13 levels were observed in WT mice receiving DSS in comparison to control. IL-13-/- mice administered DSS exhibited significantly reduced severity of colitis compared to WT mice as reflected by macroscopic and histological damage assessments. Following DSS administration, significantly lower pro-inflammatory cytokine production and fewer infiltrating macrophages were observed in IL-13-/- mice compared to WT. The reduced severity of colitis observed in IL-13-/- mice was also accompanied by down-regulation of EC cell numbers and colonic 5-HT content. In addition, increasing colonic 5-HT content by administration of rmIL-13 or 5-HTP exacerbated severity of DSS colitis in IL-13-/- mice. IL-13-/- mice also exhibited reduced severity of DNBS-induced colitis. These results demonstrate that IL-13 plays a critical role in the pathogenesis of experimental colitis and 5-HT is an important mediator of IL-13 driven intestinal inflammation. This study revealed important information on immune-endocrine axis in gut in relation to inflammation which may ultimately lead to better strategy in managing various intestinal inflammatory conditions including inflammatory bowel disease.

Proton pump inhibitors exacerbate NSAID-induced small intestinal injury by inducing dysbiosis.

BACKGROUND & AIMS: Proton pump inhibitors (PPIs) and nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used classes of drugs, with the former frequently coprescribed to reduce gastroduodenal injury caused by the latter. However, suppression of gastric acid secretion by PPIs is unlikely to provide any protection against the damage caused by NSAIDs in the more distal small intestine. METHODS: Rats were treated with antisecretory doses of omeprazole or lanzoprazole for 9 days, with concomitant treatment with anti-inflammatory doses of naproxen or celecoxib on the final 4 days. Small intestinal damage was blindly scored, and changes in hematocrit were measured. Changes in small intestinal microflora were evaluated by denaturing gradient gel electrophoresis and reverse-transcription polymerase chain reaction. RESULTS: Both PPIs significantly exacerbated naproxen- and celecoxib-induced intestinal ulceration and bleeding in the rat. Omeprazole treatment did not result in mucosal injury or inflammation; however, there were marked shifts in numbers and types of enteric bacteria, including a significant reduction (∼80%) of jejunal Actinobacteria and Bifidobacteria spp. Restoration of small intestinal Actinobacteria numbers through administration of selected (Bifidobacteria enriched) commensal bacteria during treatment with omeprazole and naproxen prevented intestinal ulceration/bleeding. Colonization of germ-free mice with jejunal bacteria from PPI-treated rats increased the severity of NSAID-induced intestinal injury, as compared with mice colonized with bacteria from vehicle-treated rats. CONCLUSIONS: PPIs exacerbate NSAID-induced intestinal damage at least in part because of significant shifts in enteric microbial populations. Prevention or reversal of this dysbiosis may be a viable option for reducing the incidence and severity of NSAID enteropathy.

The intestinal microbiota affect central levels of brain-derived neurotropic factor and behavior in mice.

BACKGROUND & AIMS: Alterations in the microbial composition of the gastrointestinal tract (dysbiosis) are believed to contribute to inflammatory and functional bowel disorders and psychiatric comorbidities. We examined whether the intestinal microbiota affects behavior and brain biochemistry in mice. METHODS: Specific pathogen-free (SPF) BALB/c mice, with or without subdiaphragmatic vagotomy or chemical sympathectomy, or germ-free BALB/c mice received a mixture of nonabsorbable antimicrobials (neomycin, bacitracin, and pimaricin) in their drinking water for 7 days. Germ-free BALB/c and NIH Swiss mice were colonized with microbiota from SPF NIH Swiss or BALB/c mice. Behavior was evaluated using step-down and light preference tests. Gastrointestinal microbiota were assessed using denaturing gradient gel electrophoresis and sequencing. Gut samples were analyzed by histologic, myeloperoxidase, and cytokine analyses; levels of serotonin, noradrenaline, dopamine, and brain-derived neurotropic factor (BDNF) were assessed by enzyme-linked immunosorbent assay. RESULTS: Administration of oral antimicrobials to SPF mice transiently altered the composition of the microbiota and increased exploratory behavior and hippocampal expression of BDNF. These changes were independent of inflammatory activity, changes in levels of gastrointestinal neurotransmitters, and vagal or sympathetic integrity. Intraperitoneal administration of antimicrobials to SPF mice or oral administration to germ-free mice did not affect behavior. Colonization of germ-free BALB/c mice with microbiota from NIH Swiss mice increased exploratory behavior and hippocampal levels of BDNF, whereas colonization of germ-free NIH Swiss mice with BALB/c microbiota reduced exploratory behavior. CONCLUSIONS: The intestinal microbiota influences brain chemistry and behavior independently of the autonomic nervous system, gastrointestinal-specific neurotransmitters, or inflammation. Intestinal dysbiosis might contribute to psychiatric disorders in patients with bowel disorders.

Host responses to intestinal microbial antigens in gluten-sensitive mice.

BACKGROUND AND AIMS: Excessive uptake of commensal bacterial antigens through a permeable intestinal barrier may influence host responses to specific antigen in a genetically predisposed host. The aim of this study was to investigate whether intestinal barrier dysfunction induced by indomethacin treatment affects the host response to intestinal microbiota in gluten-sensitized HLA-DQ8/HCD4 mice. METHODOLOGY/PRINCIPAL FINDINGS: HLA-DQ8/HCD4 mice were sensitized with gluten, and gavaged with indomethacin plus gluten. Intestinal permeability was assessed by Ussing chamber; epithelial cell (EC) ultra-structure by electron microscopy; RNA expression of genes coding for junctional proteins by Q-real-time PCR; immune response by in-vitro antigen-specific T-cell proliferation and cytokine analysis by cytometric bead array; intestinal microbiota by fluorescence in situ hybridization and analysis of systemic antibodies against intestinal microbiota by surface staining of live bacteria with serum followed by FACS analysis. Indomethacin led to a more pronounced increase in intestinal permeability in gluten-sensitized mice. These changes were accompanied by severe EC damage, decreased E-cadherin RNA level, elevated IFN-gamma in splenocyte culture supernatant, and production of significant IgM antibody against intestinal microbiota. CONCLUSION: Indomethacin potentiates barrier dysfunction and EC injury induced by gluten, affects systemic IFN-gamma production and the host response to intestinal microbiota antigens in HLA-DQ8/HCD4 mice. The results suggest that environmental factors that alter the intestinal barrier may predispose individuals to an increased susceptibility to gluten through a bystander immune activation to intestinal microbiota.

In vivo replication of T4 and T7 bacteriophages in germ-free mice colonized with Escherichia coli.

The gut transit of T4 phages was studied in axenic mice mono-colonized with the non-pathogenic Escherichia coli strain K-12. Thirty minutes, 1 and 2 h after phage feeding, T4 phage had reached the jejunum, ileum and cecum, respectively. Phage was found in the lumen and was also associated with the mucosa. One day later no phage was detected in the feces. Compared to germ-free control animals, oral T4 phage led to a 300-fold higher fecal phage titer in mice mono-colonized with E. coli strain WG-5. The in vivo T4 phage replication was transient and reached peak fecal titers about 8 h after oral phage application followed by a rapid titer decrease over two days. Similar data were obtained in mice colonized with E. coli strain Nissle. In contrast, orally applied T7 phage experienced a massive and sustained in vivo replication in mice mono-colonized with E. coli strain WG-5 irrespective whether phage or E. coli host was applied first. T7 phage replication occurred mainly in the large intestine. High titers of T7 phage and high E. coli cell counts coexisted in the feces. The observation of only 20% T7 phage-resistant fecal E. coli colonies suggests a refuge model where phage-sensitive E. coli cells are physically or physiologically protected from phage infection in the gut. The difference between T7 and T4 with respect to gut replication might partly reflect their distinct in vitro capacity to replicate on slowly growing cells.

A Mesocosm of Lactobacillus johnsonii, Bifidobacterium longum, and Escherichia coli in the mouse gut.

The relative contribution of competition and cooperation at the microbe-microbe level is not well understood for the bacteria constituting the gut microbiota. The high number and variability of human gut commensals have hampered the analysis. To get some insight into the question how so many different bacterial species can coexist in the mammalian gut, we studied the interaction between three human gut commensals (Escherichia coli K-12, Lactobacillus johnsonii NCC533, and Bifidobacterium longum NCC2705) in the intestine of gnotobiotic mice. The bacterial titers and their anatomical distribution were studied in the colonized mice. L. johnsonii achieved the highest cell counts in the stomach, while B. longum dominated the colon. The colon was also the intestinal location in which B. longum displayed the highest number of expressed genes, followed by the cecum and the small intestine. Addition of further bacterial strains led to strikingly different results. A Lactobacillus paracasei strain coexisted, while a second B. longum strain was excluded from the system. Notably, this strain lacked an operon involved in the degradation, import, and metabolism of mannosylated glycans. Subsequent introduction of the E. coli Nissle strain resulted in the elimination of L. johnsonii NCC533 and E. coli K-12, while B. longum NCC2705 showed a transient decrease in population size, demonstrating the dynamic nature of microbe-microbe interactions. The study of such simple interacting bacterial systems might help to derive some basic rules governing microbial ecology within the mammalian gut.

T4 phages against Escherichia coli diarrhea: potential and problems.

A combination of in vitro and in vivo experiments with comparative phage genomics was used for the rational design of a phage cocktail against E. coli diarrhea. Orally applied T4 coliphages representing three different subgroups (T4-, RB49- and JS98-like phages) had no negative impact on the murine gut microbiota. T4 phages were found with high titers in the cecum and colon and lower titers in the small intestine, but were not detected in the blood, liver or spleen. No adverse effects were observed after one-month exposure to phage nor were serum anti-T4 antibodies detected. T4 phages belonging to the same subgroup showed closely related genomes that differed by 12 (phage JS10 vs. JS98 reference) to 17 (phage JSE vs. RB49 reference) insertion/deletions mostly representing single small ORFs. Bioinformatic analysis did not reveal undesired genes in the T4 genomes. Sequence variability was seen over the tail fibre genes, but the variability did not correlate with phage host range. The investigated T4 phages were not only species- but also strain-specific, necessitating the use of phage cocktails consisting of 10 and 16 T4 phage isolates to cover half to two thirds of E. coli strains representing the five main pathotypes isolated from diarrhea patients.

The role of pathogenic microbes and commensal bacteria in irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) reflects several pathogenetic entities including a subgroup with low-grade colonic inflammation. We propose that pathogenic bacteria act as triggers and that disturbances of commensal bacteria maintain low-grade inflammation, that in turn leads to dysfunction in the gut or brain. METHODS: Studies were performed in mice under specific pathogen-free conditions. Visceral pain was assessed by the visceromotor response and motility was assessed by in vivo fluoroscopy and in vitro by muscle contractility. Brain chemistry was assessed by in situ hybridization and behavior by standard tests. The microbiota was monitored using 16s-based RT-PCR and DGGE. RESULTS: Mice transiently infected with the nematode Trichinella spiralis exhibited changes in motility and in visceral perception that persisted for up to 6 weeks post-infection. This was accompanied by alterations in the microbiota and an upregulation of cyclooxygenase-2 which could be reversed by treatment with anti-inflammatory agents or selected probiotics. To investigate the contribution of the microbiota, we treated mice with oral antibiotics and monitored visceral perception and behavior. Antibiotic therapy produced substantial changes in the microbiota, a small increment in inflammatory activity and an increase in substance P or pain perception. Oral, but not systemic antibiotic treatment, produced changes in brain chemistry and an increase in anxiety-like behavior. CONCLUSION: These studies provide proof of concept that pathogenic microbes can induce persistent gut dysfunction and that changes in microbial composition of the gut can maintain gut dysfunction as well as induce behavioral changes reminiscent of the psychiatric comorbidity that occurs in up to 60% of irritable bowel syndrome patients.