Systematic profiling of the chicken gut microbiome reveals dietary supplementation with antibiotics alters expression of multiple microbial pathways with minimal impact on community structure.

BACKGROUND: The emergence of antimicrobial resistance is a major threat to global health and has placed pressure on the livestock industry to eliminate the use of antibiotic growth promotants (AGPs) as feed additives. To mitigate their removal, efficacious alternatives are required. AGPs are thought to operate through modulating the gut microbiome to limit opportunities for colonization by pathogens, increase nutrient utilization, and reduce inflammation. However, little is known concerning the underlying mechanisms. Previous studies investigating the effects of AGPs on the poultry gut microbiome have largely focused on 16S rDNA surveys based on a single gastrointestinal (GI) site, diet, and/or timepoint, resulting in an inconsistent view of their impact on community composition. METHODS: In this study, we perform a systematic investigation of both the composition and function of the chicken gut microbiome, in response to AGPs. Birds were raised under two different diets and AGP treatments, and 16S rDNA surveys applied to six GI sites sampled at three key timepoints of the poultry life cycle. Functional investigations were performed through metatranscriptomics analyses and metabolomics. RESULTS: Our study reveals a more nuanced view of the impact of AGPs, dependent on age of bird, diet, and intestinal site sampled. Although AGPs have a limited impact on taxonomic abundances, they do appear to redefine influential taxa that may promote the exclusion of other taxa. Microbiome expression profiles further reveal a complex landscape in both the expression and taxonomic representation of multiple pathways including cell wall biogenesis, antimicrobial resistance, and several involved in energy, amino acid, and nucleotide metabolism. Many AGP-induced changes in metabolic enzyme expression likely serve to redirect metabolic flux with the potential to regulate bacterial growth or produce metabolites that impact the host. CONCLUSIONS: As alternative feed additives are developed to mimic the action of AGPs, our study highlights the need to ensure such alternatives result in functional changes that are consistent with site-, age-, and diet-associated taxa. The genes and pathways identified in this study are therefore expected to drive future studies, applying tools such as community-based metabolic modeling, focusing on the mechanistic impact of different dietary regimes on the microbiome. Consequently, the data generated in this study will be crucial for the development of next-generation feed additives targeting gut health and poultry production. Video Abstract.

Effects of therapeutic levels of dietary antibiotics on the cecal microbiome composition of broiler chickens.

Dietary antibiotics, including antibiotic growth promoters (AGPs), have been commonly used to improve health and growth of poultry. The present study investigated the effects of therapeutic doses of dietary antibiotics, including bacitracin methylene disalicylate (BMD), penicillin G potassium (PP) and an ionophore (salinomycin, SA), on the cecal microbiome of chickens. BMD and SA treatments were given as dietary supplements from d 1 to 35 of age. The SAPP (salinomycin+ penicillin G potassium) group was given SA as a dietary supplement from d 1 to 35 of age and PP was added to drinking water from d 19 to 24 of age to simulate common practices for control of necrotic enteritis in broilers. The cecal contents were collected from all treatment groups on d 10, 24, and 35 of age and DNA was extracted for metagenomic analysis of the cecal microbiome. The results revealed that dietary or water supplementation of therapeutic levels of antibiotics and ionophores to chickens significantly altered the cecal microbial homeostasis during different stages of the chicken life. The alpha diversity analysis showed that BMD, SA, and SAPP treatments decreased diversity and evenness of the cecal microbiome of treated chickens on d 10 of age. Species richness was also reduced on d 35 following treatment with BMD. Beta diversity analyses revealed that SAPP and BMD induced significant changes in the relative abundance of Gram-positive and -negative bacteria on d 10, while no significant differences were observed on d 24. On d 35, the non-treated control group had higher relative abundance of unclassified Gram-positive and -negative bacteria compared to SA, SAPP, and BMD treatment groups. Overall, despite their beneficial role in controlling necrotic enteritis outbreaks, the findings of this study highlight the potential negative effects of dietary supplementation of therapeutic levels of antibiotics on the gut microbiome and suggest that adjusting gut bacteria may be required to restore microbial richness and diversity of the gut microbiome following treatment with these antibiotics.

Micronutrient supplements can promote disruptive protozoan and fungal communities in the developing infant gut.

Supplementation with micronutrients, including vitamins, iron and zinc, is a key strategy to alleviate child malnutrition. However, association of gastrointestinal disorders with iron has led to ongoing debate over their administration. To better understand their impact on gut microbiota, we analyse the bacterial, protozoal, fungal and helminth communities of stool samples collected from a subset of 80 children at 12 and 24 months of age, previously enrolled into a large cluster randomized controlled trial of micronutrient supplementation in Pakistan (ClinicalTrials.gov identifier NCT00705445). We show that while bacterial diversity is reduced in supplemented children, vitamins and iron (as well as residence in a rural setting) may promote colonization with distinct protozoa and mucormycetes, whereas the addition of zinc appears to ameliorate this effect. We suggest that the risks and benefits of micronutrient interventions may depend on eukaryotic communities, potentially exacerbated by exposure to a rural setting. Larger studies are needed to evaluate the clinical significance of these findings and their impact on health outcomes.

Modeling the metabolic interplay between a parasitic worm and its bacterial endosymbiont allows the identification of novel drug targets.

The filarial nematode Brugia malayi represents a leading cause of disability in the developing world, causing lymphatic filariasis in nearly 40 million people. Currently available drugs are not well-suited to mass drug administration efforts, so new treatments are urgently required. One potential vulnerability is the endosymbiotic bacteria Wolbachia-present in many filariae-which is vital to the worm. Genome scale metabolic networks have been used to study prokaryotes and protists and have proven valuable in identifying therapeutic targets, but have only been applied to multicellular eukaryotic organisms more recently. Here, we present iDC625, the first compartmentalized metabolic model of a parasitic worm. We used this model to show how metabolic pathway usage allows the worm to adapt to different environments, and predict a set of 102 reactions essential to the survival of B. malayi. We validated three of those reactions with drug tests and demonstrated novel antifilarial properties for all three compounds.

Synthesis of glutamic acid analogs as potent inhibitors of leukotriene A4 hydrolase.

Leukotriene B(4) (LTB(4)) is a potent pro-inflammatory mediator that has been implicated in the pathogenesis of multiple diseases, including psoriasis, inflammatory bowel disease, multiple sclerosis and asthma. As a method to decrease the level of LTB(4) and possibly identify novel treatments, inhibitors of the LTB(4) biosynthetic enzyme, leukotriene A(4) hydrolase (LTA(4)-h), have been explored. Here we describe the discovery of a potent inhibitor of LTA(4)-h, arylamide of glutamic acid 4f, starting from the corresponding glycinamide 2. Analogs of 4f are then described, focusing on compounds that are both active and stable in whole blood. This effort culminated in the identification of amino alcohol 12a and amino ester 6b which meet these criteria.

In silico footprinting of ligands binding to the minor groove of DNA.

The sequence selectivity of small molecules binding to the minor groove of DNA can be predicted by "in silico footprinting". Any potential ligand can be docked in the minor groove and then moved along it using simple simulation techniques. By applying a simple scoring function to the trajectory after energy minimization, the preferred binding site can be identified. We show application to all known noncovalent binding modes, namely 1:1 ligand:DNA binding (including hairpin ligands) and 2:1 side-by-side binding, with various DNA base pair sequences and show excellent agreement with experimental results from X-ray crystallography, NMR, and gel-based footprinting.

Comparison of bioenergetic activity of primary porcine hepatocytes cultured in four different media.

Primary hepatocytes have extensively been used in biochemical, pharmacological, and physiological research. Recently, primary porcine hepatocytes have been regarded as the cells of choice for bioartificial liver support systems. The optimum culture medium for hepatocytes to be used in such devices has yet to be defined. In this study we investigated the effectiveness of four culture media in driving energy metabolism of primary porcine hepatocytes. The media selected were William's E medium, medium 1640, medium 199, and hepatocyte medium. Cells (3 x 10(10); viability 87 +/- 6%) were isolated from weanling piglets and seeded on 90-mm plates in the above media supplemented with antibiotics and hormones at a density of 8 x 10(6) viable cells per plate. Using 1H NMR spectroscopy we looked at indices of glycolysis, gluconeogenesis. ketogenesis, and ureagenesis on days 2, 4, and 6 of the experiments (n = 9). We also studied urea and albumin synthesis and total P450 content. The examined metabolic pathways of the hepatocytes were maintained by all media, although there were statistically significant differences between them. All media performed well in glycolysis, ureagenesis, and albumin synthesis. William's E medium and medium 199 outperformed the rest in gluconeogenesis. Medium 199 was best in ketogenesis. Overall, medium 199 was the best at driving energy metabolism from its constituent substrates and we think that it preferentially should be used in the culture of primary porcine hepatocytes.

Metabolic and functional protection by selective inhibition of nitric oxide synthase 2 during ischemia-reperfusion in isolated perfused hearts.

BACKGROUND: Drugs that selectively block nitric oxide synthase (NOS) 2 enzyme activity by inhibiting dimerization of NOS2 monomers have recently been developed. METHODS AND RESULTS: To investigate whether selective inhibition of NOS2 is cardioprotective, rats were pretreated for 2 days with BBS2, an inhibitor of NOS2 dimerization, at 15 mg/kg SC. Isolated buffer-perfused hearts from treated (n=9) and control (n=7) hearts were subjected to 20 minutes of ischemia followed by 60 minutes of reperfusion. NOS2 protein was upregulated in all hearts at the end of ischemia and of reperfusion; NOS2 enzyme activity was 60% lower in hearts from the treated animals. In the treated hearts, the increase in end-diastolic pressure was significantly attenuated at the end of ischemia, and the return of developed pressure at reperfusion was greater (P<0.05). Creatine kinase release at reperfusion was lower in treated hearts than in controls (P=0.02). At the end of ischemia and of reperfusion, myocardial ATP levels were significantly higher in the treated hearts than in controls (P<0.05). In the treated hearts under ischemic conditions, lactate content was higher and the lactate/pyruvate ratio was lower than in controls (P<0.05); GAPDH activity was higher; and G-3-P and aldose reductase activity were lower. At reperfusion, in the treated hearts, there was less histological damage and less apoptosis of cardiac muscle cells. CONCLUSIONS: Pretreatment with BBS2, a selective inhibitor of NOS2, improves contractile performance, preserves myocardial ATP, and reduces damage and death of cardiac myocytes during ischemia and reperfusion of isolated buffer-perfused rat hearts.

An aspirin-triggered lipoxin A4 stable analog displays a unique topical anti-inflammatory profile.

Lipoxins and 15-epi-lipoxins are counter-regulatory lipid mediators that modulate leukocyte trafficking and promote the resolution of inflammation. To assess the potential of lipoxins as novel anti-inflammatory agents, a stable 15-epi-lipoxin A(4) analog, 15-epi-16-p-fluorophenoxy-lipoxin A(4) methyl ester (ATLa), was synthesized by total organic synthesis and examined for efficacy relative to a potent leukotriene B(4) (LTB(4)) receptor antagonist (LTB(4)R-Ant) and the clinically used topical glucocorticoid methylprednisolone aceponate. In vitro, ATLa was 100-fold more potent than LTB(4)R-Ant for inhibiting neutrophil chemotaxis and trans-epithelial cell migration induced by fMLP, but was approximately 10-fold less potent than the LTB(4)R-Ant in blocking responses to LTB(4). A broad panel of cutaneous inflammation models that display pathological aspects of psoriasis, atopic dermatitis, and allergic contact dermatitis was used to directly compare the topical efficacy of ATLa with that of LTB(4)R-Ant and methylprednisolone aceponate. ATLa was efficacious in all models tested: LTB(4)/Iloprost-, calcium ionophore-, croton oil-, and mezerein-induced inflammation and trimellitic anhydride-induced allergic delayed-type hypersensitivity. ATLa was efficacious in mouse and guinea pig skin inflammation models, exhibiting dose-dependent effects on edema, neutrophil or eosinophil infiltration, and epidermal hyperproliferation. We conclude that the LXA(4) and aspirin-triggered LXA(4) pathways play key anti-inflammatory roles in vivo. Moreover, these results suggest that ATLa and related LXA(4) analogs may have broad therapeutic potential in inflammatory disorders and could provide an alternative to corticosteroids in certain clinical settings.

IL-4 dependent alternatively-activated macrophages have a distinctive in vivo gene expression phenotype.

BACKGROUND: "Alternatively-activated" macrophages are found in Th2-mediated inflammatory settings such as nematode infection and allergic pulmonary inflammation. Due in part to a lack of markers, these cells have not been well characterized in vivo and their function remains unknown. RESULTS: We have used murine macrophages elicited by nematode infection (NeM(phi)) as a source of in vivo derived alternatively activated macrophages. Using three distinct yet complementary molecular approaches we have established a gene expression profile of alternatively activated macrophages and identified macrophage genes that are regulated in vivo by IL-4. First, genes abundantly expressed were identified by an expressed sequence tag strategy. Second, an array of 1176 known mouse genes was screened for differential expression between NeM(phi) from wild type or IL-4 deficient mice. Third, a subtractive library was screened to identify novel IL-4 dependent macrophage genes. Differential expression was confirmed by real time RT-PCR analysis. CONCLUSIONS: Our data demonstrate that alternatively activated macrophages generated in vivo have a gene expression profile distinct from any macrophage population described to date. Several of the genes we identified, including those most abundantly expressed, have not previously been associated with macrophages and thus this study provides unique new information regarding the phenotype of macrophages found in Th2-mediated, chronic inflammatory settings. Our data also provide additional in vivo evidence for parallels between the inflammatory processes involved in nematode infection and allergy.

Mechanistic studies with potent and selective inducible nitric-oxide synthase dimerization inhibitors.

A series of potent and selective inducible nitric-oxide synthase (iNOS) inhibitors was shown to prevent iNOS dimerization in cells and inhibit iNOS in vivo. These inhibitors are now shown to block dimerization of purified human iNOS monomers. A 3H-labeled inhibitor bound to full-length human iNOS monomer with apparent Kd approximately 1.8 nm and had a slow off rate, 1.2 x 10(-4) x s(-1). Inhibitors also bound with high affinity to both murine full-length and murine oxygenase domain iNOS monomers. Spectroscopy and competition binding with imidazole confirmed an inhibitor-heme interaction. Inhibitor affinity in the binding assay (apparent Kd values from 330 pm to 27 nm) correlated with potency in a cell-based iNOS assay (IC50 values from 290 pm to 270 nm). Inhibitor potency in cells was not prevented by medium supplementation with l-arginine or sepiapterin, but inhibition decreased with time of addition after cytokine stimulation. The results are consistent with a mechanism whereby inhibitors bind to a heme-containing iNOS monomer species to form an inactive iNOS monomer-heme-inhibitor complex in a pterin- and l-arginine-independent manner. The selectivity for inhibiting dimerization of iNOS versus endothelial and neuronal NOS suggests that the energetics and kinetics of monomer-dimer equilibria are substantially different for the mammalian NOS isoforms. These inhibitors provide new research tools to explore these processes.