Commensal Enteric Bacteria Research Articles

Oxidative Stress in the Poultry Gut: Potential Challenges and Interventions.

The gastrointestinal tract (GIT) provides the biological environment for nutrient digestion and absorption, and protection from pathogens and toxins. Broilers are fast growing because of the great potential of intestinal epithelia for nutrient absorption, and efficient conversion of nutrient to muscle. Physiologically, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated by GIT epithelial cells either from oxygen metabolism or by enteric commensal bacteria and regulate gut health. However, increased production of ROS elevates free radical production and antioxidant insults resulting in oxidative stress. Oxidative stress in poultry GIT is derived from nutritional, environmental heat stress, and pathological factors, which alters overall performance as well as meat and egg quality. Supplementation of exogenous vitamins, antioxidants, and plant extract having antioxidant properties scavenge ROS and are beneficial in mitigating oxidative stress in the GIT. This review highlights the involvement of oxidative stress in the gastrointestinal functionality of poultry and potential intervention strategies to maintain redox balance in the GIT.

HOIL1 Is Essential for the Induction of Type I and III Interferons by MDA5 and Regulates Persistent Murine Norovirus Infection.

The linear ubiquitin chain assembly complex (LUBAC), composed of heme-oxidized IRP2 ubiquitin ligase 1 (HOIL1), HOIL1-interacting protein (HOIP), and SHANK-associated RH domain-interacting protein (SHARPIN), is a crucial regulator of multiple immune signaling pathways. In humans, HOIL1 or HOIP deficiency is associated with an immune disorder involving autoinflammation, immunodeficiency, and inflammatory bowel disease (IBD)-like symptoms. During viral infection, LUBAC is reported to inhibit the induction of interferon (IFN) by the cytosolic RNA sensor retinoic acid-inducible gene I (RIG-I). Surprisingly, we found that HOIL1 is essential for the induction of both type I and type III IFNs, as well as the phosphorylation of IFN regulatory factor 3 (IRF3), during murine norovirus (MNoV) infection in cultured dendritic cells. The RIG-I-like receptor, melanoma differentiation-associated protein 5 (MDA5), is also required for IFN induction and IRF3 phosphorylation during MNoV infection. Furthermore, HOIL1 and MDA5 were required for IFN induction after Theiler's murine encephalomyelitis virus infection and poly(I·C) transfection, but not Sendai virus or vesicular stomatitis virus infection, indicating that HOIL1 and LUBAC are required selectively for MDA5 signaling. Moreover, Hoil1-/- mice exhibited defective control of acute and persistent murine norovirus infection and defective regulation of MNoV persistence by the microbiome as also observed previously for mice deficient in interferon lambda (IFN-λ) receptor, signal transducer and activator of transcription factor 1 (STAT1), and IRF3. These data indicate that LUBAC plays a critical role in IFN induction to control RNA viruses sensed by MDA5.IMPORTANCE Human noroviruses are a leading cause of gastroenteritis throughout the world but are challenging to study in vivo and in vitro Murine norovirus (MNoV) provides a tractable genetic and small-animal model to study norovirus biology and immune responses. Interferons are critical mediators of antiviral immunity, but excessive expression can dysregulate the immune system. IFN-λ plays an important role at mucosal surfaces, including the gastrointestinal tract, and both IFN-λ and commensal enteric bacteria are important modulators of persistent MNoV infection. LUBAC, of which HOIL1 is a component, is reported to inhibit type I IFN induction after RIG-I stimulation. We show, in contrast, that HOIL1 is critical for type I and III IFN induction during infection with MNoV, a virus that preferentially activates MDA5. Moreover, HOIL1 regulates MNoV infection in vivo These data reveal distinct functions for LUBAC in these closely related signaling pathways and in modulation of IFN expression.

Identification of factors involved in Enterococcus faecalis biofilm under quercetin stress

Enterococcus faecalis is a gram positive enteric commensal bacteria or opportunistic pathogen and its infection involves biofilm formation. Quercetin, a plant origin polyphenol was found to inhibit E. faecalis biofilm. Crystal violet assay, SEM and CLSM microscopy confirmed biofilm inhibition by quercetin. Proteomics was used to elucidate the changes occurred in bacterial cell by quercetin treatment. 2D-Electrophorosis and MALDI-TOF analysis revealed that nineteen proteins were differentially expressed in quercetin treated sample. Glycolytic pathways, protein translation-elongation pathways and protein folding pathways were under differential expression after treatment. Real Time-PCR (RT-PCR) validated the proteomic data at genomic level except for the translation elongation factor G which showed opposite data to proteomics. Protein-protein interaction networks constructed using STRING 10.0 demonstrated strong connection of translation-elongation proteins with many important proteins. The results of the comparative analysis indicate that quercetin exerts its inhibitory effect by disturbing glycolytic, protein translation-elongation and protein folding pathways. This disturbs bacterial physiology and stops transition of planktonic cells to biofilm state.

Genistein induces macrophage polarization and systemic cytokine to ameliorate experimental colitis.

Mucosal changes in Crohn’s disease (CD) and ulcerative colitis (UC), two major forms of inflammatory bowel disease (IBD), are characterized by a prominent infiltration of inflammatory cells including lymphocytes, macrophages, T cells and neutrophils. The precise etiology of IBD is unknown but it involves a complex interplay of factors associated with the immune system, environment, host genotype and enteric commensal bacteria. As there is no known safe cure for IBD, natural alternative therapeutic options without side effects are urgently needed. To this end, Soy-based foods, which have been eaten for centuries in Asian countries, have potential benefits, including lowering the incidence of coronary heart disease, atherosclerosis, type-2 diabetes, allergic response, and autoimmune diseases. This study describes the effect of Soy isoflavons 4', 5, 7 Trihydroxyisoflavone (genistein) on dextran sodium sulphate (DSS) induced experimental colitis. The extent and severity of disease was analyzed through body weight, histopathological analysis, cellular immune response, systemic cytokine levels, and inflammation score using a disease activity index. Genistein treatment significantly attenuated DSS-induced colitis severity and resulted in increase in body weight, colon length and reduction in inflammation score. Genistein also skews M1 macrophages towards the M2 phenotype. Further, gen also reduced the systemic cytokine levels as compared to vehicle control. This serves as the first detailed study towards natural soya based product that shows the polarization of M1 towards M2 macrophages, and reduction of systemic cytokine in part to attenuate the colitis symptoms. Thus, our work demonstrates that genistein, a soya compound, may be useful for the treatment of IBD.

Molecular Insights into Antimicrobial Resistance Traits of Commensal Human Gut Microbiota.

Antimicrobial resistance (AMR) among bacterial species that resides in complex ecosystems is a natural phenomenon. Indiscriminate use of antimicrobials in healthcare, livestock, and agriculture provides an evolutionary advantage to the resistant variants to dominate the ecosystem. Ascendency of resistant variants threatens the efficacy of most, if not all, of the antimicrobial drugs commonly used to prevent and/or cure microbial infections. Resistant phenotype is very common in enteric bacteria. The most common mechanisms of AMR are enzymatic modifications to the antimicrobials or their target molecules. In enteric bacteria, most of the resistance traits are acquired by horizontal gene transfer from closely or distantly related bacterial population. AMR traits are generally linked with mobile genetic elements (MGEs) and could rapidly disseminate to the bacterial species through horizontal gene transfer (HGT) from a pool of resistance genes. Although prevalence of AMR genes among pathogenic bacteria is widely studied in the interest of infectious disease management, the resistance profile and the genetic traits that encode resistance to the commensal microbiota residing in the gut of healthy humans are not well-studied. In the present study, we have characterized AMR phenotypes and genotypes of five dominant commensal enteric bacteria isolated from the gut of healthy Indians. Our study revealed that like pathogenic bacteria, enteric commensals are also multidrug-resistant. The genes encoding antibiotic resistance are physically linked with MGEs and could disseminate vertically to the progeny and laterally to the distantly related microbial species. Consequently, the AMR genes present in the chromosome of commensal gut bacteria could be a potential source of resistance functions for other enteric pathogens.

F88. MANIPULATION OF THE GUT MICROBIOTA WITH A PREBIOTIC IN SCHIZOPHRENIA: A DOUBLE-BLINDED RANDOMIZED PLACEBO-CONTROLLED CROSS-OVER STUDY

BackgroundNeurocognitive impairment is increasingly recognized as a fundamental symptomatology in schizophrenia with more than 80% of patients exhibiting significant deficits, even at first episode of illness. Current pharmacotherapies do not alleviate cognitive symptoms, and often cause severe metabolic dysfunction and weight gain that readily become major health concerns. Identifying adjunctive interventions that improve cognition without disrupting the therapeutic actions of anti-psychotic medications, and can mitigate the metabolic side-effects of these drugs, would be highly beneficial to patients and improve prognosis. We have recently demonstrated that the manipulation of the rat gut microbiota with a prebiotic (dietary fibre that grows beneficial enteric bacteria) improves cognitive flexibility and prevents olanzapine-mediated weight gain. We therefore aim to explore whether these actions of the prebiotic translate to medicated stable patients with schizophrenia.MethodsA total of 40 patients with psychosis aged 18–65 will be enrolled in a 24-week maltodextrin-controlled cross-over experimental medicine study. Participants will receive either a 12-week treatment with a prebiotic (active compound) followed by a 12-week maltodextrin supplement (placebo), or in the reverse order. The order of supplements that participants receive is randomized. The primary outcome is to examine the influence of prebiotic supplementation on neurocognitive functioning, which is measured using a tablet-based neuropsychometric test battery. We will also examine the impact on clinical metabolic measures such as total weight and visceral adiposity. The concentration of immune-related serum proteins as well as neuroendocrine hormones will be evaluated. All measurements will take place at baseline, at 12-week cross-over, and at the end of the 24-week study. A within-subjects repeated measures analysis will be performed, and co-variates (gender, weight, medication) identified. This trial is registered with ClinicalTrials.gov, identifier number NCT03153046.ResultsWe have currently screened 36 patients, of whom 30 were eligible for the study (67% male). At baseline, the average age of all recruited was 36.41 ± 11.42. The overall cognitive score was -2.03 ± 0.53 where the subtests included verbal memory (29.09 ± 8.75), digit sequencing (15.41 ± 2.77), token motor (55.74 ± 24.14), semantic fluency (8.83 ± 3.46), letter fluency (11.39 ± 3.65), symbol coding (35.61 ± 9.22) and tower of London (15.1 ± 4.06). There was no difference in overall cognitive between male (14.29 ± 2.46) and female (17.22 ± 2.30) patients. However, long-term associative learning as measured by the digit sequencing subtest appeared to show a significant difference between male (14.29 ± 2.46) and female (17.22 ± 2.33; p=0.008) participants. No significant differences between clinical metabolic measures were observed in baseline BMI (32.25 ± 6.79) and abdominal obesity as measured by hip-to-waist ratio (0.94 ± 0.11). The serum concentrations of immune and endocrine markers will also be presented.DiscussionThis investigation, to our knowledge, is the first clinical study to provide medicated schizophrenia patients with a prebiotic, as a potential means of improving cognition and managing secondary metabolic dysfunction. Although a potential link between commensal enteric bacteria and schizophrenia have been suggested, earlier work with probiotics (live cultures) did not support this association. However, since the current study uses a prebiotic that proliferates multiple species of gut bacteria, it will provide more robust data that will support, or refute, the validity of manipulating the gut microbiome in the treatment of schizophrenia.

Unveiling the Mechanism of Arginine Transport through AdiC with Molecular Dynamics Simulations: The Guiding Role of Aromatic Residues

Commensal and pathogenic enteric bacteria have developed several systems to adapt to proton leakage into the cytoplasm resulting from extreme acidic conditions. One such system involves arginine uptake followed by export of the decarboxylated product agmatine, carried out by the arginine/agmatine antiporter (AdiC), which thus works as a virtual proton pump. Here, using classical and targeted molecular dynamics, we investigated at the atomic level the mechanism of arginine transport through AdiC of E. coli. Overall, our MD simulation data clearly demonstrate that global rearrangements of several transmembrane segments are necessary but not sufficient for achieving transitions between structural states along the arginine translocation pathway. In particular, local structural changes, namely rotameric conversions of two aromatic residues, are needed to regulate access to both the outward- and inward-facing states. Our simulations have also enabled identification of a few residues, overwhelmingly aromatic, which are essential to guiding arginine in the course of its translocation. Most of them belong to gating elements whose coordinated motions contribute to the alternating access mechanism. Their conservation in all known E. coli acid resistance antiporters suggests that the transport mechanisms of these systems share common features. Last but not least, knowledge of the functional properties of AdiC can advance our understanding of the members of the amino acid-carbocation-polyamine superfamily, notably in eukaryotic cells.

Enterobacter cloacae inhibits human norovirus infectivity in gnotobiotic pigs.

Human noroviruses (HuNoVs) are the leading cause of epidemic gastroenteritis worldwide. Study of HuNoV biology has been hampered by the lack of an efficient cell culture system. Recently, enteric commensal bacteria Enterobacter cloacae has been recognized as a helper in HuNoV infection of B cells in vitro. To test the influences of E. cloacae on HuNoV infectivity and to determine whether HuNoV infects B cells in vivo, we colonized gnotobiotic pigs with E. cloacae and inoculated pigs with 2.74 × 104 genome copies of HuNoV. Compared to control pigs, reduced HuNoV shedding was observed in E. cloacae colonized pigs, characterized by significantly shorter duration of shedding in post-inoculation day 10 subgroup and lower cumulative shedding and peak shedding in individual pigs. Colonization of E. cloacae also reduced HuNoV titers in intestinal tissues and in blood. In both control and E. cloacae colonized pigs, HuNoV infection of enterocytes was confirmed, however infection of B cells was not observed in ileum, and the entire lamina propria in sections of duodenum, jejunum, and ileum were HuNoV-negative. In summary, E. cloacae inhibited HuNoV infectivity, and B cells were not a target cell type for HuNoV in gnotobiotic pigs, with or without E. cloacae colonization.

Sialidases from gut bacteria: a mini-review.

Sialidases are a large group of enzymes, the majority of which catalyses the cleavage of terminal sialic acids from complex carbohydrates on glycoproteins or glycolipids. In the gastrointestinal (GI) tract, sialic acid residues are mostly found in terminal location of mucins via α2-3/6 glycosidic linkages. Many enteric commensal and pathogenic bacteria can utilize sialic acids as a nutrient source, but not all express the sialidases that are required to release free sialic acid. Sialidases encoded by gut bacteria vary in terms of their substrate specificity and their enzymatic reaction. Most are hydrolytic sialidases, which release free sialic acid from sialylated substrates. However, there are also examples with transglycosylation activities. Recently, a third class of sialidases, intramolecular trans-sialidase (IT-sialidase), has been discovered in gut microbiota, releasing (2,7-anhydro-Neu5Ac) 2,7-anydro-N-acetylneuraminic acid instead of sialic acid. Reaction specificity varies, with hydrolytic sialidases demonstrating broad activity against α2,3-, α2,6- and α2,8-linked substrates, whereas IT-sialidases tend to be specific for α2,3-linked substrates. In this mini-review, we summarize the current knowledge on the structural and biochemical properties of sialidases involved in the interaction between gut bacteria and epithelial surfaces.

Antigen sampling by intestinal M cells is the principal pathway initiating mucosal IgA production to commensal enteric bacteria.

Secretory IgA (SIgA) directed against gut resident bacteria enables the mammalian mucosal immune system to establish homeostasis with the commensal gut microbiota after weaning. Germinal centers (GCs) in Peyer's patches (PPs) are the principal inductive sites where naive B cells specific for bacterial antigens encounter their cognate antigens and receive T-cell help driving their differentiation into IgA-producing plasma cells. We investigated the role of antigen sampling by intestinal M cells in initiating the SIgA response to gut bacteria by developing mice in which receptor activator of nuclear factor-κB ligand (RANKL)-dependent M-cell differentiation was abrogated by conditional deletion of Tnfrsf11a in the intestinal epithelium. Mice without intestinal M cells had profound delays in PP GC maturation and emergence of lamina propria IgA plasma cells, resulting in diminished levels of fecal SIgA that persisted into adulthood. We conclude that M-cell-mediated sampling of commensal bacteria is a required initial step for the efficient induction of intestinal SIgA.

Scoping review to identify potential non-antimicrobial interventions to mitigate antimicrobial resistance in commensal enteric bacteria in North American cattle production systems.

A scoping review was conducted to identify modifiable non-antimicrobial factors to reduce the occurrence of antimicrobial resistance in cattle populations. Searches were developed to retrieve peer-reviewed published studies in animal, human and in vitro microbial populations. Citations were retained when modifiable non-antimicrobial factors or interventions potentially associated with antimicrobial resistance were described. Studies described resistance in five bacterial genera, species or types, and 40 antimicrobials. Modifiable non-antimicrobial factors or interventions ranged widely in type, and the depth of evidence in animal populations was shallow. Specific associations between a factor or intervention with antimicrobial resistance in a population (e.g. associations between organic systems and tetracycline susceptibility in E. coli from cattle) were reported in a maximum of three studies. The identified non-antimicrobial factors or interventions were classified into 16 themes. Most reported associations between the non-antimicrobial modifiable factors or interventions and antimicrobial resistance were not statistically significant (P > 0·05 and a confidence interval including 1), but when significant, the results were not consistent in direction (increase or decrease in antimicrobial resistance) or magnitude. Research is needed to better understand the impacts of promising modifiable factors or interventions on the occurrence of antimicrobial resistance before any recommendations can be offered or adopted.

Selecting anti-microbial treatment of aerobic vaginitis.

Aerobic vaginitis (AV) is a vaginal infectious condition which is often confused with bacterial vaginosis (BV) or with the intermediate microflora as diagnosed by Nugent's method to detect BV on Gram-stained specimens. However, although both conditions reflect a state of lactobacillary disruption in the vagina, leading to an increase in pH, BV and AV differ profoundly. While BV is a noninflammatory condition composed of a multiplex array of different anaerobic bacteria in high quantities, AV is rather sparely populated by one or two enteric commensal flora bacteria, like Streptococcus agalactiae, Staphylocuccus aureus, or Escherichia coli. AV is typically marked by either an increased inflammatory response or by prominent signs of epithelial atrophy or both. The latter condition, if severe, is also called desquamative inflammatory vaginitis. As AV is per exclusionem diagnosed by wet mount microscopy, it is a mistake to treat just vaginal culture results. Vaginal cultures only serve as follow-up data in clinical research projects and are at most used in clinical practice to confirm the diagnosis or exclude Candida infection. AV requires treatment based on microscopy findings and a combined local treatment with any of the following which may yield the best results: antibiotic (infectious component), steroids (inflammatory component), and/or estrogen (atrophy component). In cases with Candida present on microscopy or culture, antifungals must be tried first in order to see if other treatment is still needed. Vaginal rinsing with povidone iodine can provide rapid relief of symptoms but does not provide long-term reduction of bacterial loads. Local antibiotics most suitable are preferably non-absorbed and broad spectrum, especially those covering enteric gram-positive and gram-negative aerobes, like kanamycin. To achieve rapid and short-term improvement of severe symptoms, oral therapy with amoxyclav or moxifloxacin can be used, especially in deep dermal vulvitis and colpitis infections with group B streptococci or (methicillin resistant) Staphylococcus aureus. Since the latter colonizations are frequent, but seldom inflammatory infections, we in general discourage the use of oral antibiotics in women with AV. In cases with a severe atrophy component (more than 10% of epithelial cells are of the parabasal type), local estrogens can be used; and in postmenopausal or breast cancer patients with a contraindication for estrogens, even a combination of probiotics with an ultra-low dose of local estriol may be considered.

Commensal enteric bacteria lipopolysaccharide impairs host defense against disseminated Candida albicans fungal infection.

Commensal enteric bacteria maintain systemic immune responsiveness that protects against disseminated or localized infection in extra-intestinal tissues caused by pathogenic microbes. However, since shifts in infection susceptibility after commensal bacteria eradication have primarily been probed using viruses, the broader applicability to other pathogen types remains undefined. In sharp contrast to diminished antiviral immunity, we show commensal bacteria eradication bolsters protection against disseminated Candida albicans fungal infection. Enhanced antifungal immunity reflects more robust systemic expansion of Ly6GhiLy6Cint neutrophils, and their mobilization into infected tissues among antibiotic treated compared with commensal bacteria replete control mice. Reciprocally, depletion of neutrophils from expanded levels or intestinal LPS reconstitution overrides the antifungal protective benefits conferred by commensal bacteria eradication. This discordance in antifungal compared with antiviral immunity highlights intrinsic differences in how commensal bacteria control responsiveness for specific immune cell subsets because pathogen-specific CD8+ T cells that protect against viruses were suppressed similarly after C. albicans and influenza A virus infection. Thus, positive calibration of antiviral immunity by commensal bacteria is counterbalanced by restrained activation of other immune components that confer antifungal immunity.

Inflammatory Bowel Disease: The Classic Gastrointestinal Autoimmune Disease

Inflammatory bowel disease (IBD) is an idiopathic disease thought to be caused by a dysregulated immune response to host intestinal microflora. The role of genetic factors is indicated by familial clustering of cases and higher incidence in monozygotic twins. An interaction between genetic and environmental factors is thought to play a role in the pathogenesis of these disorders. Changes in diet, antibiotic use and intestinal colonization have likely contributed to increased prevalence of inflammatory bowel disease in the past century. Environmental factors or infections are thought to alter the barrier function of the epithelium, leading to loss of immune tolerance to intestinal antigens. This loss of tolerance activates dendritic cells, triggering their transport to mesenteric lymph nodes, where they promote differentiation of naïve T cells to TH-1, TH-2, TH-17 cells or T regulatory cells. Production of proinflammatory cytokines and chemokines then follows. Circulating effector and regulatory cells enter the intestine through a highly selective mechanism that involves interaction with the vascular endothelium, diapedesis through the vessel wall and migration to the lamina propria. There are several genes implicated in IBD. Mutations in certain genes can cause defective down regulation of the innate immune response, ineffective clearance of intracellular bacteria and proliferation of both luminal and mucosal-adherent commensal bacteria. IBD is a chronic relapsing inflammatory condition that is immune mediated. Results from research in animal models, human genetics, basic science and clinical trials provide evidence that it is heterogeneous, characterized by various genetic abnormalities, leading to a dysregulated and overly aggressive T cell response to commensal enteric bacteria. Different genetic abnormalities can be characterized as causing defects in mucosal barrier function, immunoregulation or bacterial clearance. Advances in our understanding of the interplay between components of innate and adaptive immune response will be central to future progress.

The carriage of antibiotic resistance by enteric bacteria from imported tokay geckos (Gekko gecko) destined for the pet trade

The emergence of antibiotic-resistant bacteria is a growing public health concern and has serious implications for both human and veterinary medicine. The nature of the global economy encourages the movement of humans, livestock, produce, and wildlife, as well as their potentially antibiotic-resistant bacteria, across international borders. Humans and livestock can be reservoirs for antibiotic-resistant bacteria; however, little is known about the prevalence of antibiotic-resistant bacteria harbored by wildlife and, to our knowledge, limited data has been reported for wild-caught reptiles that were specifically collected for the pet trade. In the current study, we examined the antibiotic resistance of lactose-positive Enterobacteriaceae isolates from wild-caught Tokay geckos (Gekko gecko) imported from Indonesia for use in the pet trade. In addition, we proposed that the conditions under which wild animals are captured, transported, and handled might affect the shedding or fecal prevalence of antibiotic resistance. In particular we were interested in the effects of density; to address this, we experimentally modified densities of geckos after import and documented changes in antibiotic resistance patterns. The commensal enteric bacteria from Tokay geckos (G. gecko) imported for the pet trade displayed resistance against some antibiotics including: ampicillin, amoxicillin/clavulanic acid, cefoxitin, chloramphenicol, kanamycin and tetracycline. There was no significant difference in the prevalence of antibiotic-resistant bacteria after experimentally mimicking potentially stressful transportation conditions reptiles experience prior to purchase. There were, however, some interesting trends observed when comparing Tokay geckos housed individually and those housed in groups. Understanding the prevalence of antibiotic resistant commensal enteric flora from common pet reptiles is paramount because of the potential for humans exposed to these animals to acquire antibiotic-resistant bacteria and the potential for released pets to disseminate these bacteria to native wildlife.

Pseudomonas aeruginosa promotes Escherichia coli biofilm formation in nutrient-limited medium.

Biofilms have been implicated as an important reservoir for pathogens and commensal enteric bacteria such as Escherichia coli in natural and engineered water systems. However, the processes that regulate the survival of E. coli in aquatic biofilms have not been thoroughly studied. We examined the effects of hydrodynamic shear and nutrient concentrations on E. coli colonization of pre-established Pseudomonas aeruginosa biofilms, co-inoculation of E. coli and P. aeruginosa biofilms, and P. aeruginosa colonization of pre-established E. coli biofilms. In nutritionally-limited R2A medium, E. coli dominated biofilms when co-inoculated with P. aeruginosa, and successfully colonized and overgrew pre-established P. aeruginosa biofilms. In more enriched media, P. aeruginosa formed larger clusters, but E. coli still extensively overgrew and colonized the interior of P. aeruginosa clusters. In mono-culture, E. coli formed sparse and discontinuous biofilms. After P. aeruginosa was introduced to these biofilms, E. coli growth increased substantially, resulting in patterns of biofilm colonization similar to those observed under other sequences of organism introduction, i.e., E. coli overgrew P. aeruginosa and colonized the interior of P. aeruginosa clusters. These results demonstrate that E. coli not only persists in aquatic biofilms under depleted nutritional conditions, but interactions with P. aeruginosa can greatly increase E. coli growth in biofilms under these experimental conditions.

Commensal microbes drive intestinal inflammation by IL-17–producing CD4 + T cells through ICOSL and OX40L costimulation in the absence of B7-1 and B7-2

The costimulatory B7-1 (CD80)/B7-2 (CD86) molecules, along with T-cell receptor stimulation, together facilitate T-cell activation. This explains why in vivo B7 costimulation neutralization efficiently silences a variety of human autoimmune disorders. Paradoxically, however, B7 blockade also potently moderates accumulation of immune-suppressive regulatory T cells (Tregs) essential for protection against multiorgan systemic autoimmunity. Here we show that B7 deprivation in mice overrides the necessity for Tregs in averting systemic autoimmunity and inflammation in extraintestinal tissues, whereas peripherally induced Tregs retained in the absence of B7 selectively mitigate intestinal inflammation caused by Th17 effector CD4(+) T cells. The need for additional immune suppression in the intestine reflects commensal microbe-driven T-cell activation through the accessory costimulation molecules ICOSL and OX40L. Eradication of commensal enteric bacteria mitigates intestinal inflammation and IL-17 production triggered by Treg depletion in B7-deficient mice, whereas re-establishing intestinal colonization with Candida albicans primes expansion of Th17 cells with commensal specificity. Thus, neutralizing B7 costimulation uncovers an essential role for Tregs in selectively averting intestinal inflammation by Th17 CD4(+) T cells with commensal microbe specificity.

Gastrointestinal Microbes Interact with Canine Adipose-Derived Mesenchymal Stem Cells In Vitro and Enhance Immunomodulatory Functions

Mesenchymal stem cells (MSCs) are somatic, multipotent stromal cells with potent immunomodulatory and regenerative properties. Although MSCs have pattern recognition receptors and are modulated by Toll-like receptor ligands, MSC-microbial interactions are poorly defined. The objectives of this study were to determine the effect of bacterial association on MSC function. We hypothesized that gastrointestinal bacteria associate with MSCs and alter their immunomodulatory properties. The effect of MSC-microbial interactions on MSC morphology, viability, proliferation, migration, and immunomodulatory functions was investigated. MSCs associated with a remarkable array of enteric pathogens and commensal bacteria. MSC interactions with two model organisms, the pathogen Salmonella typhimurium and the probiotic Lactobacillus acidophilus, were further investigated. While ST readily invaded MSCs, LB adhered to the MSC plasma membrane. Neither microbe induced MSC death, degeneration, or diminished proliferation. Microbial association did not upregulate MHC-II, CD80/86, or CD1 expression. MSC-microbial interaction significantly increased transcription of key immunomodulatory genes, including COX2, IL6, and IL8, coupled with significantly increased prostaglandin E2 (PGE2), interleukin (IL)6, and IL8 secretion. MSC-ST coincubation resulted in increased MSC expression of CD54, and significant augmentation of MSC inhibition of mitogen-induced T-cell proliferation. T-cell proliferation was partially restored when PGE2 secretion was blocked from ST-primed MSCs. MSC-microbe interactions have a profound effect on MSC function and may be pivotal in a variety of clinical settings where MSCs are being explored as potential therapeutics in the context of microbial communities, such as Crohn's disease, chronic nonhealing wounds, and sepsis.

Enteric commensal bacteria activate formyl peptide receptor 1 to regulate intestinal epithelial homeostasis and wound repair (488.7)

Mucosal homeostasis and wound repair require epithelial proliferation and migration. Gut commensal microbiota are essential for normal intestinal development, renewal and repair. N‐formyl peptide receptors (FPRs) are G protein coupled receptors that function as pattern recognition receptors. FPR ligands include an array of peptides, lipids and small molecules derived from bacteria and the host. Intestinal epithelial cells (IECs) express functional FPRs in the cell surface plasma membrane. Recently we demonstrated that Annexin A1 and specific gut microbiota stimulate epithelial FPRs to generate reactive oxygen species which modulate cell signaling pathways critical for IEC proliferation and migration. However, it is incompletely understood how FPR1 maintains homeostasis of the gut symbionts important for mucosal wound healing. Here, we studied composition of murine mucosa‐associated microbiota in intact epithelia and endoscopic wound beds by pyrosequencing. We identified that specific species of essential gut bacteria is associated with mucosal wound bed. We also observed that FPR1 maintains homeostasis of mucosa‐associated microbiota characterized by their enrichment, composition, and spatial localization with respect to IECs. These changes are also associated with alterations in the colonic firm mucin layer, increased epithelial permeability and defective mucosal wound healing in FPR1‐/‐ mice during chronic DSS‐induced colitis. In conclusion, our study highlights a role of FPR1 in mediating important host‐microbiota interactions controlling gut mucosal homeostasis and wound repair.Grant Funding Source: CCFA and NIH

Intestinal microbiota, probiotics and prebiotics in inflammatory bowel disease.

It has been presumed that aberrant immune response to intestinal microorganisms in genetically predisposed individuals may play a major role in the pathogenesis of the inflammatory bowel disease, and there is a good deal of evidence supporting this hypothesis. Commensal enteric bacteria probably play a central role in pathogenesis, providing continuous antigenic stimulation that causes chronic intestinal injury. A strong biologic rationale supports the use of probiotics and prebiotics for inflammatory bowel disease therapy. Many probiotic strains exhibit anti-inflammatory properties through their effects on different immune cells, pro-inflammatory cytokine secretion depression, and the induction of anti-inflammatory cytokines. There is very strong evidence supporting the use of multispecies probiotic VSL#3 for the prevention or recurrence of postoperative pouchitis in patients. For treatment of active ulcerative colitis, as well as for maintenance therapy, the clinical evidence of efficacy is strongest for VSL#3 and Escherichia coli Nissle 1917. Moreover, some prebiotics, such as germinated barley foodstuff, Psyllium or oligofructose-enriched inulin, might provide some benefit in patients with active ulcerative colitis or ulcerative colitis in remission. The results of clinical trials in the treatment of active Crohn's disease or the maintenance of its remission with probiotics and prebiotics are disappointing and do not support their use in this disease. The only exception is weak evidence of advantageous use of Saccharomyces boulardii concomitantly with medical therapy in maintenance treatment.