Organ growth and fermentation profiles of broilers differing in body growth rate.

This study sought to determine the relationship among broiler performance, organ development, and indicators of microbiota colonization. A total of 1,200 two-day-old male Ross 308 broiler chicks, divided among 3 cohorts of equal size, were housed in battery cages, and allotted based on body weight. On study d 11, birds were weighed, and birds with BW gain within the 10th and 90th percentiles were assigned to the Slow and Fast groups, respectively. Birds (n = 30 for each group) selected on d 11 were provided water and a corn-soybean meal-based diet ad libitum while maintained individually through study d 25 (i.e., a 14-d growth period). Parameters regarding growth performance, organ and intestine weights and lengths, and intestinal volatile fatty acid concentrations were measured. All data were analyzed by one-way ANOVA using the Mixed procedure of SAS. Fast birds exhibited greater (P < 0.001) BW gain and feed intake than slow birds, but feed conversion ratio (FCR) did not differ (P = 0.19). Additionally, Slow birds had higher (P < 0.05) relative weights (% of BW) for nearly all organs on d 11 and 25, most notably the gizzard, proventriculus, pancreas, and liver. Conversely, intestinal sections were longer (P < 0.05) in the Fast birds. Measurement of gut histomorphology did not show any notable differences between growth rate groups in terms of villi height, crypt depth, or their ratio for either time-point (P > 0.05). In terms of volatile fatty acid concentrations of luminal contents, acetate concentrations were 10.2% higher (P < 0.001) in the ileum of the Slow birds compared with Fast birds on d 25. Overall, the findings suggest that total BW gain is influenced by the development of metabolically active organs, as supported by lower weight gain in Slow birds with relatively larger organ weights and shorter intestinal lengths than their Fast counterparts. The general lack of differences in fermentation end-product concentrations in luminal contents does not rule out influence of the microbiota on growth rate of broilers, which warrants further investigation.

Survey of clinical and commensal Escherichia coli from commercial broilers and turkeys, with emphasis on high-risk clones using APECTyper.

Molecular characterization of avian pathogenic Escherichia coli (APEC) is challenging due to the complex nature of its associated disease, colibacillosis, in poultry. Numerous efforts have been made toward defining APEC, and it is becoming clear that certain clonal backgrounds are predictive of an avian E. coli isolate's virulence potential. Thus, APEC can be further differentiated as high-risk APEC based upon their clonal background's virulence potential. However, less clear is the degree of overlap between clinical isolates of differing bird type, and between clinical and gastrointestinal isolates. This study aimed to determine genomic similarities and differences between such populations, comparing commercial broiler vs. turkey isolates, and clinical vs. gastrointestinal isolates. Differences were observed in Clermont phylogenetic groups between isolate populations, with B2 as the dominant group in turkey clinical isolates and G as the dominant group in broiler clinical isolates. Nearly all clinical isolates were classified as APEC using a traditional gene-based typing scheme, whereas 53.4% and 44.1% of broiler and turkey gastrointestinal isolates were classified as APEC, respectively. High-risk APEC were identified among 31.0% and 46.9% of broiler and turkey clinical isolates, compared with 5.7% and 2.9% of broiler and turkey gastrointestinal isolates. As found in previous studies, no specific known virulence or fitness gene sets were identified which universally differentiate between clinical and gastrointestinal isolates. This study further demonstrates the utility of a hybrid APEC typing approach, considering both plasmid content and clonal background, for the identification of dominant and highly virulent APEC clones in poultry production.

Dose-Dependent Effects of Supplementing a Two-Strain Bacillus subtilis Probiotic on Growth Performance, Blood Parameters, Fecal Metabolites, and Microbiome in Nursery Pigs.

This experiment was conducted to evaluate the effects of dietary supplementation level of a two-strain Bacillus subtilis probiotic on growth performance, blood parameters, fecal metabolites, and microbiome in nursery pigs. A total of 54 weaned piglets were allotted to three treatments in three replicate pens with six pigs/pen for a 28 d feeding trial. The treatments were as follows: control: no probiotic supplementation; Pro1x: B. subtilis supplementation at 1.875 × 105 CFU/g diet; and Pro10x: B. subtilis supplementation at 1.875 × 106 CFU/g diet. Body weight at d 14 postweaning (p = 0.06) and average daily gain for d 0 to 14 postweaning (p < 0.05) were greater in the Pro1x treatment than in the other treatments. Blood glucose levels were greater in both probiotic treatments than in the control treatment at d 14 postweaning (p < 0.05). In the fecal short-chain fatty acid (SCFA) concentrations, the butyrate concentrations were greater in the Pro1x treatment than in the other treatments (p < 0.05), and the acetate, propionate, and total SCFA concentrations were greater in the Pro1x treatment than in the Pro10x treatment (p < 0.05). The beta diversity of fecal microbiome composition at d 14 postweaning based on Unweighted Unifrac analysis was dissimilar between the Pro1x and Pro10x treatments (p < 0.05). In conclusion, dietary B. subtilis supplementation of two strains selected to reduce effects of pathogenic Escherichia coli to nursery diets at 1.875 × 105 CFU/g diet improved the growth rate in the early postweaning period, increased fecal SCFA concentrations and altered the fecal microbial community composition. A higher dose of B. subtilis did not improve the performance parameters over those of the control piglets.

Gut Microbiota-Derived Indole-3-Carboxylate Influences Mucosal Integrity and Immunity Through the Activation of the Aryl Hydrocarbon Receptors and Nutrient Transporters in Broiler Chickens Challenged With Eimeria maxima.

Two studies were conducted to evaluate the effects of indole-3-carboxylate (ICOOH) as a postbiotic on maintaining intestinal homeostasis against avian coccidiosis. In the first study, an in vitro culture system was used to investigate the effects of ICOOH on the proinflammatory cytokine response of chicken macrophage cells (CMCs), gut integrity of chicken intestinal epithelial cells (IECs), differentiation of quail muscle cells (QMCs), and primary chicken embryonic muscle cells (PMCs) and anti-parasitic effect against Eimeria maxima. Cells to be tested were seeded in the 24-well plates and treated with ICOOH at concentrations of 0.1, 1.0, and 10.0 µg. CMCs were first stimulated by lipopolysaccharide (LPS) to induce an innate immune response, and QMCs and PMCs were treated with 0.5% and 2% fetal bovine serum, respectively, before they were treated with ICOOH. After 18 h of incubation, cells were harvested, and RT-PCR was performed to measure gene expression of proinflammatory cytokines of CMCs, tight junction (TJ) proteins of IECs, and muscle cell growth markers of QMCs and PMCs. In the second study, in vivo trials were carried out to study the effect of dietary ICOOH on disease parameters in broiler chickens infected with E. maxima. One hundred twenty male broiler chickens (0-day-old) were allocated into the following four treatment groups: 1) basal diet without infection (CON), 2) basal diet with E. maxima (NC), 3) ICOOH at 10.0 mg/kg feed with E. maxima (HI), and 4) ICOOH at 1.0 mg/kg feed with E. maxima (LO). Body weights (BWs) were measured on 0, 7, 14, 20, and 22 days. All groups except the CON chickens were orally infected with E. maxima on day 14. Jejunal samples were collected for lesion score and the transcriptomic analysis of cytokines and TJ proteins. In vitro, ICOOH increased the expression of TJ proteins in IECs and decreased IL-1ß and IL-8 transcripts in the LPS-stimulated CMCs. In vivo, chickens on the HI diet showed reduced jejunal IL-1ß, IFN-γ, and IL-10 expression and increased expression of genes activated by aryl hydrocarbon receptors and nutrient transporters in E. maxima-infected chickens. In conclusion, these results demonstrate the beneficial effects of dietary ICOOH on intestinal immune responses and barrier integrity in broiler chickens challenged with E. maxima. Furthermore, the present finding supports the notion to use microbial metabolites as novel feed additives to enhance resilience in animal agriculture.

Effects of Dietary Maltol on Innate Immunity, Gut Health, and Growth Performance of Broiler Chickens Challenged With Eimeria maxima.

Two studies were conducted to evaluate the effects of maltol as a postbiotic on innate immunity, gut health, and enteric infection. In the first study, an in vitro culture system was used to evaluate the effects of maltol on the innate immune response of chicken macrophage cells (CMC), gut integrity of chicken intestinal epithelial cells (IEC), anti-parasitic activity against Eimeria maxima, and differentiation of quail muscle cells (QMC) and primary chicken embryonic muscle cells (PMC). All cells seeded in the 24-well plates were treated with maltol at concentrations of 0.1, 1.0, and 10.0 µg. CMC and IEC were stimulated by lipopolysaccharide to induce an innate immune response, and QMC and PMC were treated with 0.5 and 2% fetal bovine serum, respectively. After 18 h of incubation, pro-inflammatory cytokines, tight junction proteins (TJPs), and muscle cell growth markers were measured. In the second study, the dietary effect of maltol was evaluated on disease parameters in broiler chickens infected with E. maxima. Eighty male 1-day-old broiler chickens were allocated into the following four treatment groups: (1) Control group without infection, (2) Basal diet with E. maxima, (3) High maltol (HI; 10.0 mg /kg feed) with E. maxima, and (4) Low maltol (LO; 1.0 mg/kg feed) with E. maxima. Body weights (BW) were measured on days 0, 7, 14, 20, and 22. All chickens except the CON group were orally infected with 104 E. maxima per chicken on day 14. Jejunum samples were collected for gut lesion scoring, and the gene expression of cytokines and TJPs. Data was analyzed using PROC MIXED in SAS. In vitro, maltol not only increased TJPs in IEC and cytokines in the LPS-stimulated CMC but also showed direct cytotoxicity against sporozoites of E. maxima. In vivo, the HI group improved the BW, reduced the gut lesion scores and fecal oocyst shedding, and decreased jejunal TNFSF15 and IL-1ß expression in E. maxima-infected chickens. In conclusion, these results demonstrate the beneficial effects of dietary maltol in the enhancement of growth performance, gut health, and coccidiosis resistance and the applicability of maltol as a postbiotic for the replacement of antibiotic growth promoters in commercial poultry production.

Comparative Genomics of Clostridium perfringens Reveals Patterns of Host-Associated Phylogenetic Clades and Virulence Factors.

Clostridium perfringens is an opportunistic pathogenic bacterium that infects both animals and humans. Clostridium perfringens genomes encode a diverse array of toxins and virulence proteins, which continues to expand as more genomes are sequenced. In this study, the genomes of 44 C. perfringens strains isolated from intestinal sections of diseased cattle and from broiler chickens from diseased and healthy flocks were sequenced. These newly assembled genomes were compared to 141 publicly available C. perfringens genome assemblies, by aligning known toxin and virulence protein sequences in the assemblies using BLASTp. The genes for alpha toxin, collagenase, a sialidase (nanH), and alpha-clostripain were present in at least 99% of assemblies analyzed. In contrast, beta toxin, epsilon toxin, iota toxin, and binary enterotoxin of toxinotypes B, C, D, and E were present in less than 5% of assemblies analyzed. Additional sequence variants of beta2 toxin were detected, some of which were missing the leader or signal peptide sequences and therefore likely not secreted. Some pore-forming toxins involved in intestinal diseases were host-associated, the netB gene was only found in avian isolates, while netE, netF, and netG were only present in canine and equine isolates. Alveolysin was positively associated with canine and equine strains and only present in a single monophyletic clade. Strains from ruminant were not associated with known virulence factors and, except for the food poisoning associated clade, were present across the phylogenetic diversity identified to date for C. perfringens. Many C. perfringens strains associated with food poisoning lacked the genes for hyaluronidases and sialidases, important for attaching to and digesting complex carbohydrates found in animal tissues. Overall, the diversity of virulence factors in C. perfringens makes these species capable of causing disease in a wide variety of hosts and niches.

Dietary Supplementation With Bacillus subtilis Direct-Fed Microbials Alters Chicken Intestinal Metabolite Levels.

Direct-fed microbials (DFMs) are dietary supplements containing live microorganisms which confer a performance and health benefit to the host, but the mechanisms are unclear. Here, a metabolomics approach was used to identify changes in intestinal metabolite levels in chickens fed an unsupplemented diet or a diet supplemented with B. subtilis strain 1781 or strain 747. Body weight gains of chickens fed the B. subtilis-supplemented diets were increased up to 5.6% in the B. subtilis 1781 group and 7.6% in the B. subtilis 747 group compared with chickens fed the unsupplemented diet. Compared with unsupplemented controls, the levels of 83 metabolites were altered (p < 0.05) (25 increased, 58 decreased) in chickens given the B. subtilis 1781-supplemented diet, while 50 were altered (p < 0.05) (12 increased, 38 decreased) with the B. subtilis 747-supplemented diet. Twenty-two metabolites were altered (p < 0.05) (18 increased, 4 decreased) in the B. subtilis 1781 vs. B. subtilis 747 groups. A random forest analysis of the B. subtilis 1781 vs. control groups gave a predictive accuracy of 87.5%, while that of the B. subtilis 747 vs. control groups was 62.5%. A random forest analysis of the B. subtilis 1781 vs. B. subtilis 747 groups gave a predictive accuracy of 75.0%. Changes in the levels of these intestinal biochemicals provided a distinctive biochemical signature unique to each B. subtilis-supplemented group, and were characterized by alterations in the levels of dipeptides (alanylleucine, glutaminylleucine, phenylalanylalanine, valylglutamine), nucleosides (N1-methyladenosine, N6-methyladenosine, guanine, 2-deoxyguanosine), fatty acids (sebacate, valerylglycine, linoleoylcholine), and carbohydrates (fructose). These results provide the foundation for future studies to identify biochemicals that might be used to improve poultry growth performance in the absence of antibiotic growth promoters.

A direct comparison of endothelial progenitor cell dysfunction in rat metabolic syndrome and diabetes.

OBJECTIVES: Diabetes mellitus (DM) is associated with impairment of endothelial progenitor cells (EPCs), but the effects of metabolic syndrome (MS) on EPCs have been less well characterized. We hypothesized that in the presence of MS, the number and functionality of EPCs would be markedly reduced, and would be similar to DM. METHODS: Mononuclear cells were isolated from the bone-marrow (BM) and peripheral blood of lean Zucker, obese Zucker, a model of MS, and Zucker diabetic fatty rats. Cultured BM-EPCs underwent in vitro functional testing and the ability of BM-EPCs to promote neovascularization in vivo was assessed in a model of hindlimb ischemia in athymic mice. RESULTS: While circulating EPC numbers were similarly reduced in both MS and DM rats, BM-derived EPC numbers were less affected. In vitro testing of cultured BM-EPCs from obese Zucker demonstrated a marked reduction in EPC differentiation, a greater propensity to apoptosis, a reduced migratory response and matrigel tubule formation, similar to findings in Zucker diabetic fatty rats. When delivered to the ischemic hindlimb of athymic mice, the recovery of perfusion using both BM-EPCs from obese Zucker and Zucker diabetic fatty rats were diminished, as compared to lean Zuckers. CONCLUSION: In the presence of the MS, BM-derived EPCs develop marked functional impairment, resulting in severely reduced angiogenic capacity in vivo. Similar to DM, EPC dysfunction may play a prominent role in the pathogenesis of vascular complications in the MS, and may potentially limit the use of BM-derived EPCs for therapeutic angiogenesis.

Sustained improvement in perfusion and flow reserve after temporally separated delivery of vascular endothelial growth factor and angiopoietin-1 plasmid deoxyribonucleic acid.

OBJECTIVES: The aim of this study was to compare temporally separated vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-1 delivery with concomitant delivery or single VEGF delivery, for therapeutic angiogenesis in chronic ischemia. BACKGROUND: Single gene delivery of VEGF results in immature neovessels that ultimately regress. Endogenously, VEGF acts early to initiate angiogenesis, whereas Ang-1 acts later to induce vessel maturation. Timing VEGF and Ang-1 gene delivery to mimic endogenous angiogenesis might be more effective for sustained neovascularization. METHODS: Unilateral hindlimb ischemia was induced in 170 rats. Ultrasound-mediated gene delivery was performed with cationic microbubbles and plasmid deoxyribonucleic acid. Groups included VEGF at 2 weeks, VEGF/Ang-1 at 2 weeks, VEGF at 2 weeks with Ang-1 at 4 weeks, and untreated control subjects. At 2, 4, and 8 weeks after ligation, blood flow and flow reserve (FR) were assessed by contrast-enhanced ultrasound. Vascular density, organization, and supporting cell coverage were assessed by fluorescent microangiography and immunohistochemistry. RESULTS: In untreated control subjects, blood flow, FR, and vessel density remained reduced. The VEGF delivery improved flow and vessel density at 4 weeks; however, FR remained low, supporting cell coverage was poor, and flow and vessel density regressed by 8 weeks. The VEGF/Ang-1 co-delivery marginally increased flow and vessel density; however, FR and supporting cell coverage improved. After temporally separated VEGF and Ang-1 delivery, blood flow, vessel density, and FR increased and were sustained, with improved pericyte coverage at 8 weeks. CONCLUSIONS: In conclusion, temporally separated VEGF and Ang-1 gene therapy results in sustained and functional neovascularization.

Contrast ultrasound and targeted microbubbles: diagnostic and therapeutic applications for angiogenesis.

Angiogenesis represents the formation of new capillaries from existing vasculature, and as such plays a critical role in the response to ischemia in the setting of chronic coronary artery and peripheral vascular disease. Recent technological advances in non-invasive imaging modalities now allow the molecular imaging of angiogenesis. One such technique is contrast-enhanced ultrasound using microbubbles targeted against molecular markers of the angiogenic process. The ability to non-invasively image the angiogenic process would be useful in risk stratifying patients with arterial occlusive disease and would aid in the evaluation of new therapies to promote angiogenesis in ischemic cardiac and skeletal muscle. Furthermore, ultrasound technologies have also been developed that allow targeted angiogenic gene therapy using high-power ultrasound and DNA-bearing microbubbles. This review will focus specifically on recent advances in (1) contrast-enhanced ultrasound molecular imaging techniques for the evaluation of angiogenesis and (2) ultrasound-mediated gene delivery for therapeutic angiogenesis, techniques that have potential for translation to clinical practice.

Quantification of Propionibacterium acidipropionici P169 bacteria in environmental samples by use of strain-specific primers derived by suppressive subtractive hybridization.

A quantitative PCR (qPCR) assay targeting a gene identified by suppressive subtractive hybridization (SSH) was developed to detect Propionibacterium acidipropionici P169, with a threshold of 10(4) CFU/U of dairy feed or rumen fluid. The report is the first using a molecular marker generated by SSH to quantify a bacterial strain in environmental samples.

Stem Cell Therapy: A New Treatment for Burns?

Stem cell therapy has emerged as a promising new approach in almost every medicine specialty. This vast, heterogeneous family of cells are now both naturally (embryonic and adult stem cells) or artificially obtained (induced pluripotent stem cells or iPSCs) and their fates have become increasingly controllable, thanks to ongoing research in this passionate new field. We are at the beginning of a new era in medicine, with multiple applications for stem cell therapy, not only as a monotherapy, but also as an adjunct to other strategies, such as organ transplantation or standard drug treatment. Regrettably, serious preclinical concerns remain and differentiation, cell fusion, senescence and signalling crosstalk with growth factors and biomaterials are still challenges for this promising multidisciplinary therapeutic modality. Severe burns have several indications for stem cell therapy, including enhancement of wound healing, replacement of damaged skin and perfect skin regeneration - incorporating skin appendages and reduced fibrosis -, as well as systemic effects, such as inflammation, hypermetabolism and immunosuppression. The aim of this review is to describe well established characteristics of stem cells and to delineate new advances in the stem cell field, in the context of burn injury and wound healing.

Molecular imaging of endothelial progenitor cell engraftment using contrast-enhanced ultrasound and targeted microbubbles.

AIMS: Imaging methods to track the fate of progenitor cells after their delivery would be useful in assessing the efficacy of cell-based therapies. We hypothesized that contrast-enhanced ultrasound (CEU) using microbubbles targeted to a genetically engineered cell-surface marker on endothelial progenitor cells (EPCs) would allow the targeted imaging of vascular engraftment. METHODS AND RESULTS: Rodent bone marrow-derived EPCs were isolated, cultured, and transfected to express the marker protein, H-2Kk, on the cell surface. Non-transfected EPCs and EPCs transfected with either null plasmid or Firefly luciferase served as controls. Control microbubbles (MB(C)) and microbubbles targeted to H-2Kk expressed on EPCs (MB(H-2Kk)) were constructed. Binding of targeted microbubbles to EPCs was assessed in vitro using a parallel plate flow chamber system. CEU imaging of EPC-targeted microbubbles was assessed in vivo using subcutaneously implanted EPC-supplemented Matrigel plugs in rats. In flow chamber experiments, there was minimal attachment of microbubbles to plated control EPCs. Although numbers of adhered MB(C) were also low, there was greater and more diffuse attachment of MB(H-2Kk) to plated H-2Kk-transfected EPCs. Targeted CEU demonstrated marked contrast enhancement at the periphery of the H-2Kk-transfected EPC-supplemented Matrigel plug for MB(H-2Kk,) whereas contrast enhancement was low for MB(C). Contrast enhancement was also low for both microbubbles within control mock-transfected EPC plugs. The signal intensity within the H-2Kk-transfected EPC plug was significantly greater for MB(H-2Kk) when compared with MB(C). CONCLUSION: Microbubbles targeted to a genetically engineered cell-surface marker on EPCs exhibit specific binding to EPCs in vitro. These targeted microbubbles bind to engrafted EPCs in vivo within Matrigel plugs and can be detected by their enhancement on CEU imaging.

The BaeSR two-component regulatory system mediates resistance to condensed tannins in Escherichia coli.

The gene expression profiles of Escherichia coli strains grown anaerobically with or without Acacia mearnsii (black wattle) extract were compared to identify tannin resistance strategies. The cell envelope stress protein gene spy and the multidrug transporter-encoding operon mdtABCD, both under the control of the BaeSR two-component regulatory system, were significantly up-regulated in the presence of tannins. BaeSR mutants were more tannin sensitive than their wild-type counterparts.

Adaptation of a luciferase gene reporter and lac expression system to Borrelia burgdorferi.

The development of new genetic systems for studying the complex regulatory events that occur within Borrelia burgdorferi is an important goal of contemporary Lyme disease research. Although recent advancements have been made in the genetic manipulation of B. burgdorferi, there still remains a paucity of basic molecular systems for assessing differential gene expression in this pathogen. Herein, we describe the adaptation of two powerful genetic tools for use in B. burgdorferi. The first is a Photinus pyralis firefly luciferase gene reporter that was codon optimized to enhance translation in B. burgdorferi. Using this modified reporter, we demonstrated an increase in luciferase expression when B. burgdorferi transformed with a shuttle vector encoding the outer surface protein C (OspC) promoter fused to the luciferase reporter was cultivated in the presence of fresh rabbit blood. The second is a lac operator/repressor system that was optimized to achieve the tightest degree of regulation. Using the aforementioned luciferase reporter, we assessed the kinetics and maximal level of isopropyl-beta-D-thiogalactopyranoside (IPTG)-dependent gene expression. This lac-inducible expression system also was used to express the gene carried on lp25 required for borrelial persistence in ticks (bptA). These advancements should be generally applicable for assessing further the regulation of other genes potentially involved in virulence expression by B. burgdorferi.

Evidence that RpoS (sigmaS) in Borrelia burgdorferi is controlled directly by RpoN (sigma54/sigmaN).

The alternative sigma factor (RpoN-RpoS) pathway controls the expression of key virulence factors in Borrelia burgdorferi. However, evidence to support whether RpoN controls rpoS directly or, perhaps, indirectly via a transactivator has been lacking. Herein we provide biochemical and genetic evidence that RpoN directly controls rpoS in B. burgdorferi.

Bacterial mechanisms to overcome inhibitory effects of dietary tannins.

High concentrations of tannins in fodder plants inhibit gastrointestinal bacteria and reduce ruminant performance. Increasing the proportion of tannin-resistant bacteria in the rumen protects ruminants from anti-nutritional effects. The reason for the protective effect is unclear, but could be elucidated if the mechanism(s) by which tannins inhibit bacteria and the mechanisms of tannin resistance were understood. A review of the literature indicates that the ability of tannins to complex with polymers and minerals is the basis of the inhibitory effect on gastrointestinal bacteria. Mechanisms by which bacteria can overcome inhibition include tannin modification/degradation, dissociation of tannin-substrate complexes, tannin inactivation by high-affinity binders, and membrane modification/repair and metal ion sequestration. Understanding the mechanism of action of tannins and the mechanism(s) bacteria use to overcome the inhibitory effects will allow better management of the rumen ecosystem to reduce the anti-nutritional effects of tannin-rich fodder plants and thereby improve ruminant production.

Effect of condensed tannins on bacterial diversity and metabolic activity in the rat gastrointestinal tract.

The effect of dietary condensed tannins (proanthocyanidins) on rat fecal bacterial populations was ascertained in order to determine whether the proportion on tannin-resistant bacteria increased and if there was a change in the predominant bacterial populations. After 3 weeks of tannin diets the proportion of tannin-resistant bacteria increased significantly (P < 0.05) from 0.3% +/- 5.5% to 25.3% +/- 8.3% with a 0.7% tannin diet and to 47.2% +/- 5.1% with a 2% tannin diet. The proportion of tannin-resistant bacteria returned to preexposure levels in the absence of dietary tannins. A shift in bacterial populations was confirmed by molecular fingerprinting of fecal bacterial populations by denaturing gradient gel electrophoresis (DGGE). Posttreatment samples were generally still distinguishable from controls after 3.5 weeks. Sequence analysis of DGGE bands and characterization of tannin-resistant isolates indicated that tannins selected for Enterobacteriaceae and Bacteroides species. Dot blot quantification confirmed that these gram-negative bacterial groups predominated in the presence of dietary tannins and that there was a corresponding decrease in the gram-positive Clostridium leptum group and other groups. Metabolic fingerprint patterns revealed that functional activities of culturable fecal bacteria were affected by the presence of tannins. Condensed tannins of Acacia angustissima altered fecal bacterial populations in the rat gastrointestinal tract, resulting in a shift in the predominant bacteria towards tannin-resistant gram-negative Enterobacteriaceae and Bacteroides species.

Increasing the oxidative stress response allows Escherichia coli to overcome inhibitory effects of condensed tannins.

Tannins are plant-derived polyphenols with antimicrobial effects. The mechanism of tannin toxicity towards Escherichia coli was determined by using an extract from Acacia mearnsii (Black wattle) as a source of condensed tannins (proanthocyanidins). E. coli growth was inhibited by tannins only when tannins were exposed to oxygen. Tannins auto-oxidize, and substantial hydrogen peroxide was generated when they were added to aerobic media. The addition of exogenous catalase permitted growth in tannin medium. E. coli mutants that lacked HPI, the major catalase, were especially sensitive to tannins, while oxyR mutants that constitutively overexpress antioxidant enzymes were resistant. A tannin-resistant mutant was isolated in which a promoter-region point mutation increased the level of HPI by 10-fold. Our results indicate that wattle condensed tannins are toxic to E. coli in aerobic medium primarily because they generate H(2)O(2). The oxidative stress response helps E. coli strains to overcome their inhibitory effect.