Commensal Enteric Bacteria Research Articles

The roles of ABCB1/P-glycoprotein drug transporters in regulating gut microbes and inflammation: insights from animal models, old and new.

Commensal enteric bacteria have evolved systems that enable growth in the ecologic niche of the host gastrointestinal tract. Animals evolved parallel mechanisms to survive the constant exposure to bacteria and their metabolic by-products. We propose that drug transporters encompass a crucial system to managing the gut microbiome. Drug transporters are present in the apical surface of gut epithelia. They detoxify cells from small molecules and toxins (xenobiotics) in the lumen. Here, we review what is known about commensal structure in the absence of the transporter ABCB1/P-glycoprotein in mammalian models. Knockout or low-activity alleles of ABCB1 lead to dysbiosis, Crohn's disease and ulcerative colitis in mammals. However, the exact function of ABCB1 in these contexts remain unclear. We highlight emerging models-the zebrafish Danio rerio and sea urchin Lytechinus pictus-that are poised to help dissect the fundamental mechanisms of ATP-binding cassette (ABC) transporters in the tolerance of commensal and pathogenic communities in the gut. We and others hypothesize that ABCB1 plays a direct role in exporting inflammatory bacterial products from host epithelia. Interdisciplinary work in this research area will lend novel insight to the transporter-mediated pathways that impact microbiome community structure and accelerate the pathogenesis of inflammatory bowel disease when perturbed. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

Nutrition of Escherichia coli within the intestinal microbiome.

In this chapter, we update our 2004 review of "The Life of Commensal Escherichia coli in the Mammalian Intestine" (https://doi.org/10.1128/ecosalplus.8.3.1.2), with a change of title that reflects the current focus on "Nutrition of E. coli within the Intestinal Microbiome." The earlier part of the previous two decades saw incremental improvements in understanding the carbon and energy sources that E. coli and Salmonella use to support intestinal colonization. Along with these investigations of electron donors came a better understanding of the electron acceptors that support the respiration of these facultative anaerobes in the gastrointestinal tract. Hundreds of recent papers add to what was known about the nutrition of commensal and pathogenic enteric bacteria. The fact that each biotype or pathotype grows on a different subset of the available nutrients suggested a mechanism for succession of commensal colonizers and invasion by enteric pathogens. Competition for nutrients in the intestine has also come to be recognized as one basis for colonization resistance, in which colonized strain(s) prevent colonization by a challenger. In the past decade, detailed investigations of fiber- and mucin-degrading anaerobes added greatly to our understanding of how complex polysaccharides support the hundreds of intestinal microbiome species. It is now clear that facultative anaerobes, which usually cannot degrade complex polysaccharides, live in symbiosis with the anaerobic degraders. This concept led to the "restaurant hypothesis," which emphasizes that facultative bacteria, such as E. coli, colonize the intestine as members of mixed biofilms and obtain the sugars they need for growth locally through cross-feeding from polysaccharide-degrading anaerobes. Each restaurant represents an intestinal niche. Competition for those niches determines whether or not invaders are able to overcome colonization resistance and become established. Topics centered on the nutritional basis of intestinal colonization and gastrointestinal health are explored here in detail.

Evaluating Antimicrobial Resistance Trends in Commensal Escherichia coli Isolated from Cecal Samples of Swine at Slaughter in the United States, 2013-2019.

The emergence of antimicrobial resistance (AMR) in commensal and pathogenic enteric bacteria of swine is a public health threat. This study evaluated publicly available AMR surveillance data collected by the National Antimicrobial Resistance Monitoring System (NARMS) by assessing AMR patterns and temporal trends in commensal E. coli isolated from cecal samples of swine at slaughter across the United States. We applied the Mann-Kendall test (MKT) and a linear regression trend line to detect significant trends in the proportion of resistant isolates to individual antimicrobials over the study period. A Poisson regression model assessed differences among years in the number of antimicrobials to which an E. coli isolate was resistant. Among the 3237 E. coli isolates, a very high prevalence of resistance for tetracycline (67.62%), and high resistance for streptomycin (24.13%), and ampicillin (21.10%) were identified. The MKT and the linear trend line showed a significantly increasing temporal trend for amoxicillin-clavulanic acid, ampicillin, azithromycin, cefoxitin, ceftriaxone, and trimethoprim-sulfamethoxazole. Compared to 2013 the number of antimicrobials to which an E. coli isolate was resistant was significantly higher in the years 2017, 2018, and 2019. The increasing temporal trend of resistance to important antimicrobials for human medicine (e.g., third-generation cephalosporins) and the increase in multidrug resistance in the later years of the study are concerning and should be followed up by studies to identify sources and risk factors for the selection of AMR.

Effects of chronic stress and intestinal inflammation on commercial poultry health and performance: A review

The gastrointestinal tract provides the biological environment for nutrient digestion and absorption. Its physical and chemical barriers are crucial to protect from invading pathogens and toxic substances. On this basis, the intactness of the gastrointestinal tract, with its multiple functions and impacts, is one of the key prerequisites for human and animal health. Undoubtedly, the functions of a healthy gut system also largely benefit the welfare and performance of animals in farming systems such as poultry industries. Broiler chickens grow rapidly, as a result of rigorous genetic programs, due to the high absorption capacity of intestinal epithelia for nutrients, the quick transport of nutrients to the muscle, and their efficient conversion into energy and biomass. Due to oxygen metabolism or enteric commensal bacteria, intestinal epithelial cells create reactive oxygen and nitrogen species physiologically. However, increased generation of these oxidants goes along with the formation of free radicals resulting in oxidative stress causing lipid peroxidation and dramatic molecular changes in the structure and function of the cell and mitochondrial membranes. These effects contribute to chronic oxidative stress and inflammation of the gastrointestinal tract and generally affect all chicken organs, tissues, and cells. Hence, all forms of chronic stress, regardless of the origin, negatively impact the chicken's overall performance, health, and welfare. This review article highlights some enteric inflammation models and biomarkers to evaluate gut integrity in chickens and discusses the repercussions that chronic stress and intestinal inflammation have on the health and performance of commercial poultry

Conjugative RP4 Plasmid-Mediated Transfer of Antibiotic Resistance Genes to Commensal and Multidrug-Resistant Enteric Bacteria In Vitro

Many antibiotic-resistant bacteria carry resistance genes on conjugative plasmids that are transferable to commensals and pathogens. We determined the ability of multiple enteric bacteria to acquire and retransfer a broad-host-range plasmid RP4. We used human-derived commensal Escherichia coli LM715-1 carrying a chromosomal red fluorescent protein gene and green fluorescent protein (GFP)-labeled broad-host-range RP4 plasmid with ampR, tetR, and kanR in in vitro matings to rifampicin-resistant recipients, including Escherichia coli MG1655, Dec5α, Vibrio cholerae, Pseudomonas putida, Pseudomonas aeruginosa, Klebsiella pneumoniae, Citrobacter rodentium, and Salmonella Typhimurium. Transconjugants were quantified on selective media and confirmed using fluorescence microscopy and PCR for the GFP gene. The plasmid was transferred from E. coli LM715-1 to all tested recipients except P. aeruginosa. Transfer frequencies differed between specific donor-recipient pairings (10-2 to 10-8). Secondary retransfer of plasmid from transconjugants to E. coli LM715-1 occurred at frequencies from 10-2 to 10-7. A serial passage plasmid persistence assay showed plasmid loss over time in the absence of antibiotics, indicating that the plasmid imposed a fitness cost to its host, although some plasmid-bearing cells persisted for at least ten transfers. Thus, the RP4 plasmid can transfer to multiple clinically relevant bacterial species without antibiotic selection pressure.

Transmissible Gastroenteritis Virus Infection Promotes the Self-Renewal of Porcine Intestinal Stem Cells via Wnt/β-Catenin Pathway.

Intestinal stem cells (ISCs) play an important role in tissue repair after injury. A recent report delineates the effect of transmissible gastroenteritis virus (TGEV) infection on the small intestine of recovered pigs. However, the mechanism behind the epithelium regeneration upon TGEV infection remains unclear. To address this, we established a TGEV infection model based on the porcine intestinal organoid monolayer. The results illustrated that the porcine intestinal organoid monolayer was susceptible to TGEV. In addition, the TGEV infection initiated the interferon and inflammatory responses following the loss of absorptive enterocytes and goblet cells. However, TGEV infection did not disturb epithelial integrity but induced the proliferation of ISCs. Furthermore, TGEV infection activated the Wnt/β-catenin pathway by upregulating the accumulation and nuclear translocation of β-catenin, as well as promoting the expression of Wnt target genes, such as C-myc, Cyclin D1, Mmp7, Lgr5, and Sox9, which were associated with the self-renewal of ISCs. Collectively, these data demonstrated that the TGEV infection activated the Wnt/β-catenin pathway to promote the self-renewal of ISCs and resulted in intestinal epithelium regeneration. IMPORTANCE The intestinal epithelium is a physical barrier to enteric viruses and commensal bacteria. It plays an essential role in maintaining the balance between the host and intestinal microenvironment. In addition, intestinal stem cells (ISCs) are responsible for tissue repair after injury. Therefore, prompt self-renewal of intestinal epithelium will facilitate the rebuilding of the physical barrier and maintain gut health. In the manuscript, we found that the transmissible gastroenteritis virus (TGEV) infection did not disturb epithelial integrity but induced the proliferation of ISCs and facilitated epithelium regeneration. Detailed mechanism investigations revealed that the TGEV infection activated the Wnt/β-catenin pathway to promote the self-renewal of ISCs and resulted in intestinal epithelium regeneration. These findings will contribute to understanding the mechanism of intestinal epithelial regeneration and reparation upon viral infection.

C4-Dicarboxylates as Growth Substrates and Signaling Molecules for Commensal and Pathogenic Enteric Bacteria in Mammalian Intestine.

The C4-dicarboxylates (C4-DC) l-aspartate and l-malate have been identified as playing an important role in the colonization of mammalian intestine by enteric bacteria, such as Escherichia coli and Salmonella enterica serovar Typhimurium, and succinate as a signaling molecule for host-enteric bacterium interaction. Thus, endogenous and exogenous fumarate respiration and related functions are required for efficient initial growth of the bacteria. l-Aspartate represents a major substrate for fumarate respiration in the intestine and a high-quality substrate for nitrogen assimilation. During nitrogen assimilation, DcuA catalyzes an l-aspartate/fumarate antiport and serves as a nitrogen shuttle for the net uptake of ammonium only, whereas DcuB acts as a redox shuttle that catalyzes the l-malate/succinate antiport during fumarate respiration. The C4-DC two-component system DcuS-DcuR is active in the intestine and responds to intestinal C4-DC levels. Moreover, in macrophages and in mice, succinate is a signal that promotes virulence and survival of S. Typhimurium and pathogenic E. coli. On the other hand, intestinal succinate is an important signaling molecule for the host and activates response and protective programs. Therefore, C4-DCs play a major role in supporting colonization of enteric bacteria and as signaling molecules for the adaptation of host physiology.

Foraging risk in scavenging ecology: A study of scavenger behavior and patterns of bacterial growth

Scavenging constitutes an understudied energy pathway in terrestrial ecosystems, with important connections to disease ecology. A prevailing null hypothesis in scavenging ecology is that carcasses serve as a risk-free meal for whatever animal first encounters them on the landscape. We tested this hypothesis by focusing on a suspected risk that scavengers would face at carcasses: the risk of pathogen exposure. We conducted field trials with mouse carcasses in which we manipulated potential cues to pathogen risk and then monitored scavenger foraging decisions. Separately, we studied pathogen and commensal bacteria dynamics within mouse carcasses through time in the laboratory to better understand how carcass age might impact pathogen risk to scavengers. A visual cue to pathogen risk in the field (carcasses deployed in groups of 7 in 1 m2) caused facultative vertebrate scavengers to pass on the opportunity to feed at a rate six-times higher than for carcasses deployed singly (46.9% vs. 7.7%), suggesting an ability to perceive cues to pathogen risk at carcasses. The cues to carcass age, however, produced no effect on facultative scavenger behavior in our field trials. Laboratory trials demonstrated that both commensal enteric bacteria and a known pathogen (Listeria monocytogenes) increased at least through bloat and active decay stages in carcasses, suggesting that cues to carcass age may not help scavengers reduce pathogen risk early in decomposition. In providing evidence counter to the free meal hypothesis, our results support the continued formation of an alternative risk-based framework to understand scavenger behavior at carcasses.

Granzyme B+ CD4 T cells accumulate in the colon during chronic HIV-1 infection

ABSTRACT Chronic HIV-1 infection results in the sustained disruption of gut homeostasis culminating in alterations in microbial communities (dysbiosis) and increased microbial translocation. Major questions remain on how interactions between translocating microbes and gut immune cells impact HIV-1-associated gut pathogenesis. We previously reported that in vitro exposure of human gut cells to enteric commensal bacteria upregulated the serine protease and cytotoxic marker Granzyme B (GZB) in CD4 T cells, and GZB expression was further increased in HIV-1-infected CD4 T cells. To determine if these in vitro findings extend in vivo, we evaluated the frequencies of GZB+ CD4 T cells in colon biopsies and peripheral blood of untreated, chronically infected people with HIV-1 (PWH). Colon and blood GZB+ CD4 T cells were found at significantly higher frequencies in PWH. Colon, but not blood, GZB+ CD4 T cell frequencies were associated with gut and systemic T cell activation and Prevotella species abundance. In vitro, commensal bacteria upregulated GZB more readily in gut versus blood or tonsil-derived CD4 T cells, particularly in inflammatory T helper 17 cells. Bacteria-induced GZB expression in gut CD4 T cells required the presence of accessory cells, the IL-2 pathway and in part, MHC Class II. Overall, we demonstrate that GZB+ CD4 T cells are prevalent in the colon during chronic HIV-1 infection and may emerge following interactions with translocated bacteria in an IL-2 and MHC Class II-dependent manner. Associations between GZB+ CD4 T cells, dysbiosis and T cell activation suggest that GZB+ CD4 T cells may contribute to gut HIV-1 pathogenesis.

Potent bactericidal activity of reduced cryptdin-4 derived from its hydrophobicity and mediated by bacterial membrane disruption.

Defensin is a cysteine-rich antimicrobial peptide with three disulphide bonds under normal oxidative conditions. Cryptdin-4 (Crp4) is a defensin secreted by Paneth cells in the small intestine of mice, and only reduced Crp4 (Crp4red) shows activity against enteric commensal bacteria, although both oxidised Crp4 (Crp4ox) and Crp4red can kill non-commensal bacteria. To investigate the molecular factors that affect the potent antimicrobial activity of Crp4red, the bactericidal activities of Crp4ox and Crp4red, Crp4 with all Cys residues substituted with Ser peptide (6C/S-Crp4), and Crp4 with all thiol groups modified by N-ethylmaleimide (NEM-Crp4) were assessed. All peptides showed bactericidal activity against non-commensal bacteria, whereas Crp4red and NEM-Crp4 showed bactericidal activity against commensal bacteria. These potent peptides exhibited high hydrophobicity, which was strongly correlated with membrane insertion. Intriguingly, Crp4ox formed electrostatic interactions with the membrane surface of bacteria, even without exerting bactericidal activity. Moreover, the bactericidal activity of both oxidised and reduced forms of Crp4 was abolished by inhibition of electrostatic interactions; this finding suggests that Crp4red targets bacterial membranes. Finally, a liposome leakage assay against lipids extracted from commensal bacteria demonstrated a correlation with bactericidal activity. These results suggest that the potent bactericidal activity of Crp4red is derived from its hydrophobicity, and the bactericidal mechanism involves disruption of the bacterial membrane. Findings from this study provide a better understanding of the bactericidal mechanism of both Crp4ox and Crp4red.

P294 AMT-101, a gut selective oral IL-10 fusion: results from a Phase 1b study in patients with active Ulcerative Colitis

Abstract Background IL-10 is a pleiotropic anti-inflammatory cytokine that restores mucosal homeostasis. Genetic deficiencies in IL-10 cause intestinal inflammation in mice and are associated with early onset IBD in humans. In mice, this can be reversed by addition of exogenous IL-10. Subcutaneous use of recombinant human IL-10 treatment in patients with Crohn’s Disease led to symptomatic and endoscopic improvement, but dose limiting systemic toxicity including anemia and thrombocytopenia was observed and appeared to be mediated by induction of gamma-interferon. AMT-101 is an oral, gut selective, fusion protein of IL-10 and a carrier protein that mediates transcytosis through intestinal enterocytes into the lamina propria (LP). Pre-clinical studies demonstrated that AMT-101 efficiently transits the epithelial cell barrier, targets mucosal immune cells in the LP, and results in improved colitis disease severity without systemic exposure. Methods A multiple ascending dose Ph1b POC trial was conducted in 16 patients with active UC where patients were randomized 3:1 to receive AMT-101 at doses of 1, 3, 10, or 30 mg or PBO, orally, once daily for 14 days. Primary and secondary outcome measures included safety and PK. Exploratory endpoints included changes in fecal calprotectin, histology, and stool microbiome. Results AMT-101 was safe and well tolerated; all AEs were mild to moderate and self limiting. No systemic toxicities associated with prior administration of IL-10 (e.g.anemia or thrombocytopenia) were observed. Systemic levels of AMT-101 were below limit of quantitation, given the GI restricted design. 1 mg and 3 mg AMT-101 led to placebo-adjusted mean reductions of FCP of 44% and 27%, respectively, in patients with baseline FCP>150 μg/g. Paired biopsy samples were centrally read in a blinded manner using the Geboes scoring system. Improvements were observed in 60% (6/10) of patients on AMT-101, compared with 0% (0/2) patients treated with placebo. Microbiome analyses revealed changes in phyla abundance in the 1mg and 3mg treatment cohorts (Bacteroidetes: 28.3% ± 31.4 to 51.3% ± 21.6; Firmicutes: 21.8% ± 12.7 to 30% ± 7.3), including Roseburia hominis and Faecalibacterium prausnitzii species. Conclusion The results of this Phase 1b study confirm that once daily, oral AMT-101 was safe and well tolerated without SAEs previously observed with systemic IL-10 and without systemic exposure, by design. Doses at 10 mg or less suggest potential clinical efficacy paired with an enhancement of favorable enteric commensal bacteria after only 14 days of treatment. These findings support AMT-101 as actively exerting an immunomodulatory effect in the intestinal lamina propria and support ongoing Ph2 trials of AMT-101.

Microbiome or Infections: Amyloid-Containing Biofilms as a Trigger for Complex Human Diseases.

The human microbiota is the community of microorganisms that live upon or within their human host. The microbiota consists of various microorganisms including bacteria, fungi, viruses, and archaea; the gut microbiota is comprised mostly of bacteria. Many bacterial species within the gut microbiome grow as biofilms, which are multicellular communities embedded in an extracellular matrix. Studies have shown that the relative abundances of bacterial species, and therefore biofilms and bacterial byproducts, change during progression of a variety of human diseases including gastrointestinal, autoimmune, neurodegenerative, and cancer. Studies have shown the location and proximity of the biofilms within the gastrointestinal tract might impact disease outcome. Gram-negative enteric bacteria secrete the amyloid curli, which makes up as much as 85% of the extracellular matrix of enteric biofilms. Curli mediates cell-cell attachment and attachment to various surfaces including extracellular matrix components such as fibronectin and laminin. Structurally, curli is strikingly similar to pathological and immunomodulatory human amyloids such as amyloid-β, which has been implicated in Alzheimer's disease, α-synuclein, which is involved in Parkinson's disease, and serum amyloid A, which is secreted during the acute phase of inflammation. The immune system recognizes both bacterial amyloid curli and human amyloids utilizing the same receptors, so curli also induces inflammation. Moreover, recent work indicates that curli can participate in the self-assembly process of pathological human amyloids. Curli is found within biofilms of commensal enteric bacteria as well as invasive pathogens; therefore, evidence suggests that curli contributes to complex human diseases. In this review, we summarize the recent findings on how bacterial biofilms containing curli participate in the pathological and immunological processes in gastrointestinal diseases, systemic autoimmune diseases, and neurodegenerative diseases.

The Immunopathogenesis of Alzheimer's Disease Is Related to the Composition of Gut Microbiota.

The microbiota–gut–brain axis plays an important role in the development of neurodegenerative diseases. Commensal and pathogenic enteric bacteria can influence brain and immune system function by the production of lipopolysaccharides and amyloid. Dysbiosis of the intestinal microbiome induces local and consecutively systemic immune-mediated inflammation. Proinflammatory cytokines then trigger neuroinflammation and finally neurodegeneration. Immune-mediated oxidative stress can lead to a deficiency of vitamins and essential micronutrients. Furthermore, the wrong composition of gut microbiota might impair the intake and metabolization of nutrients. In patients with Alzheimer’s disease (AD) significant alterations of the gut microbiota have been demonstrated. Standard Western diet, infections, decreased physical activity and chronic stress impact the composition and diversity of gut microbiota. A higher abundancy of “pro-inflammatory” gut microbiota goes along with enhanced systemic inflammation and neuroinflammatory processes. Thus, AD beginning in the gut is closely related to the imbalance of gut microbiota. Modulation of gut microbiota by Mediterranean diet, probiotics and curcumin can slow down cognitive decline and alter the gut microbiome significantly. A multi-domain intervention approach addressing underlying causes of AD (inflammation, infections, metabolic alterations like insulin resistance and nutrient deficiency, stress) appears very promising to reduce or even reverse cognitive decline by exerting positive effects on the gut microbiota.

Effects of Zinc and Menthol-Based Diets on Co-Selection of Antibiotic Resistance among E. coli and Enterococcus spp. in Beef Cattle.

Simple SummaryAs antibiotic resistance increases globally, alternatives to antibiotics are increasingly being investigated as growth promoters, as well as preventive and therapeutic agents, particularly in agriculture. Equally important is the need for investigation into the effects of antibiotic alternatives on antibiotic resistance and particularly their risk for co-selection. In this study, we explored the prevalence of antibiotic-resistant Escherichia coli and Enterococcus spp. in cattle fed zinc, menthol or a combination of the two. We found that zinc supplementation was associated with higher levels of macrolide resistance observed among enterococcal isolates.Antibiotic resistance represents a growing crisis in both human and veterinary medicine. We evaluated the use of antibiotic alternatives—heavy metals and essential oils—in beef cattle feeding, and their effects on Gram-negative and Gram-positive bacteria. In this randomized controlled field trial, we measured the impact of supplemental zinc and menthol on antibiotic resistance among commensal enteric bacteria of feeder cattle. Fecal suspensions were plated onto plain- and antibiotic-supplemented MacConkey and m-Enterococcus agar for quantification of total and antibiotic-resistant Escherichia coli and Enterococcus spp., respectively. Temporal effects on overall E. coli growth were significant (p < 0.05), and menthol was associated with decreased growth on tetracycline-supplemented agar. Zinc was associated with significant increases in growth on erythromycin-supplemented m-Enterococcus agar. Cattle fed zinc exhibited significantly higher levels of macrolide resistance among fecal enterococci isolates.

Iron Reshapes the Gut Microbiome and Host Metabolism.

Compelling studies have established that the gut microbiome is a modifier of metabolic health. Changes in the composition of the gut microbiome are influenced by genetics and the environment, including diet. Iron is a potential node of crosstalk between the host-microbe relationship and metabolic disease. Although iron is well characterized as a frequent traveling companion of metabolic disease, the role of iron is underappreciated because the mechanisms of iron's influence on host metabolism are poorly characterized. Both iron deficiency and excessive amounts leading to iron overload can have detrimental effects on cardiometabolic health. Optimal iron homeostasis is critical for regulation of host immunity and metabolism in addition to regulation of commensal and pathogenic enteric bacteria. In this article we review evidence to support the notion that altering composition of the gut microbiome may be an important route via which iron impacts cardiometabolic health. We discuss reshaping of the microbiome by iron, the physiological significance and the potential for therapeutic interventions.

4412 Unraveling the role of the interaction between enteric virus and commensal bacteria in a physiological relevant model of human intestinal epithelium

OBJECTIVES/GOALS: In the crowded environment of the intestine, selected commensal bacteria and enteric viruses interact. The biological significance of this interaction, in either normal or pathological condition is not known. To study this interaction, we are developing a physiologically relevant model of an human intestinal epithelium. METHODS/STUDY POPULATION: Intestinal biopsies (ileum region) and fecal samples of 6 healthy and 6 active Crohn’s patients are being collected to derive human intestinal enteroid (HIE) lines. 2D-polarized HIE will be first characterized with studies of epithelial permeability, tight junctions and cell type composition, and co-cultured with matching fecal samples. The (co-)cultures will be then infected with human norovirus (HNoV), our model enteric virus, and infection will be quantified by RT-qPCR. In addition, the interaction of HNoV with bacteria derived from healthy or Crohn’s will be determined quantitatively by flow cytometry (viral tagging) and qualitatively by 16S sequencing of the total versus HNoV-bound bacterial species. RESULTS/ANTICIPATED RESULTS: Crohn’s patients are characterized by a microbiome dysbiosis and, in particular, by a high abundance of Enterobacteriacae. HNoV interacts with Enterobacter cloacae, and interestingly, HNoV infection is associated with exacerbation and reactivation of Crohn’s disease. By re-creating the intestinal milieu of healthy and Crohn’s patients, we expect that the kinetics of infection by HNoV will be higher in Crohn’s as compared to healthy volunteers. In addition, by studying the composition of the HNoV-bound bacterial component of Crohn’s versus heathy volunteers, we will be able to identify the contribution of selected bacteria to the expected increase of infection. DISCUSSION/SIGNIFICANCE OF IMPACT: With this study, we will fill the gap of knowledge on the importance of commensal bacteria and enteric virus interactions in healthy and diseased condition. This new knowledge will be paramount for the identification of novel strategies to combat highly prevalent virus infections.

Macrolide-susceptible probiotic Enterococcus faecium ST296 exhibits faecal-environmental-oral microbial community cycling among beef cattle in feedlots.

Enterococci are included in the United States National Antimicrobial Resistance Monitoring System to track antibiotic resistance among commensal Gram-positive enteric bacteria, largely due to their high abundance in food animals and in retail meat. In the U.S. cattle industry, macrolides are used to prevent and control liver abscesses, which cause significant economic losses. Previous studies have suggested that feeding tylosin and the intensity of the pen environment, both expand and sustain respectively the prevalence of multidrug resistance among enterococci in feedlot cattle. This has led to research into alternative feed supplements and improved stewardship practices. In a randomized controlled trial, we measured the impact of a probiotic and an altered pen environment on antimicrobial resistance among faecal Enterococcus spp. in cattle fed tylosin. Supplementing cattle with an Enterococcus faecium and Saccharomyces cerevisiae-based probiotic yielded the isolation of E. faecium of the probiotic sequence type (ST296) from faecal and environmental samples in treatment groups, as well as from cattle and the manure pack in nearby pens. Of importance, the probiotic strain also was found in a desiccated and milled manure pack sample taken 120days after the initial trial ended. Phylogenetic and SNP analyses revealed clonal survival and spread compatible with faecal-environmental-oral recycling of the probiotic strain within and among cattle and pens. The increase in prevalence of the ST296 strain occurred concomitant with a decrease in ST240, the dominant sequence type associated with ermB and tet(M) resistance genes in this trial. SIGNIFICANCE AND IMPACT OF THE STUDY: We demonstrate that a macrolide-susceptible probiotic Enterococcus faecium ST296 strain fed to beef cattle becomes fully embedded in the microbial community cycling of bacteria via faecal-environmental-oral transmission within and among feedlot pens. An initial investment in feeding the probiotic is thereby leveraged into expanding numbers of susceptible bacteria in cattle and their environment, even among those cattle fed tylosin.

Antimicrobial resistance among commensal enteric bacteria isolated from healthy cattle in Libya

Introduction: Enterococcus and E. coli are important commensals bacteria widely used as bio-indicators to monitor the occurrence and distribution of antimicrobial resistance.

Enteric bacteria induce IFNγ and Granzyme B from human colonic Group 1 Innate Lymphoid Cells

ABSTRACT Group 1 Innate Lymphoid Cells (which include Natural Killer cells and ILC1s) aid in gut anti-bacterial defense through the production of IFNγ, which is critical for mobilizing protective responses against enteric pathogens. When intestinal epithelial barrier integrity is compromised, commensal bacteria are likely to translocate from the gut lumen into the lamina propria. Few studies have addressed the mechanisms by which commensal bacteria impact the function of gut Group 1 ILCs, especially ILC1s. Utilizing an in vitro human colonic lamina propria mononuclear cell (LPMC) model, we evaluated Group 1 ILC cytokine and cytolytic protein production in response to a panel of enteric Gram-positive and Gram-negative commensal and pathogenic bacteria. IFNγ-production by NK cells and ILC1s was significantly increased after LPMC exposure to Gram-negative commensal or pathogenic bacteria, but not after exposure to the Gram-positive bacteria commensals tested. Stimulation of IFNγ production from Group 1 ILCs was not through direct recognition of bacteria by NK cells or ILC1s, but rather required accessory cells within the LPMC population. Myeloid dendritic cells generated IL-12p70, IL-18, and IL-1β upon exposure to enteric bacteria and these cytokines contributed to Group 1 ILC production of IFNγ. Furthermore, Gram-negative commensal or pathogenic bacteria induced significant expression of Granzyme B in NK cells and ILC1s. Overall, these data demonstrate that some enteric commensal bacteria indirectly induce inflammatory cytokine production and cytolytic protein expression from human colonic Group 1 ILCs, a process which could contribute to inflammation in the setting of microbial translocation.

Commensal and Pathogenic Bacteria Indirectly Induce IL-22 but Not IFNγ Production From Human Colonic ILC3s via Multiple Mechanisms.

Innate lymphoid cells (ILCs) are a diverse family of cells that play critical roles in mucosal immunity. One subset of the ILC family, Group 3 ILCs (ILC3s), has been shown to aid in gut homeostasis through the production of IL-22. IL-22 promotes gut homeostasis through its functional effect on the epithelial barrier. When gut epithelial barrier integrity is compromised, such as in Human Immunodeficiency Virus (HIV) infection and inflammatory bowel disease (IBD), microbes from the gut lumen translocate into the lamina propria, inducing a multitude of potentially pathogenic immune responses. In murine models of bacterial infection, there is evidence that bacteria can induce pro-inflammatory IFNγ production in ILC3s. However, the impact of diverse translocating bacteria, particularly commensal bacteria, in dictating IFNγ versus IL-22 production by human gut ILC3s remains unclear. Here, we utilized an in vitro human lamina propria mononuclear cell (LPMC) model to evaluate ILC3 cytokine production in response to a panel of enteric Gram-positive and Gram-negative commensal and pathogenic bacteria and determined potential mechanisms by which these cytokine responses were induced. The percentages of IL-22-producing ILC3s, but not IFNγ-producing ILC3s, were significantly increased after LPMC exposure to both Gram-positive and Gram-negative commensal or pathogenic bacterial stimuli. Stimulation of IL-22 production from ILC3s was not through direct recognition of bacterial antigen by ILC3s, but rather required the help of accessory cells within the LPMC population. CD11c+ myeloid dendritic cells generated IL-23 and IL-1β in response to enteric bacteria and contributed to ILC3 production of IL-22. Furthermore, ligation of the natural cytotoxicity receptor NKp44 on ILC3s in response to bacteria stimulation also significantly increased the percentage of IL-22-producing ILC3s. Overall, these data demonstrate that human gut microbiota, including commensal bacteria, indirectly modulate colonic ILC3 function to induce IL-22, but additional signals are likely required to induce IFNγ production by colonic ILC3s in the setting of inflammation and microbial translocation.