33 research outputs found

    A Product of Heme Catabolism Modulates Bacterial Function and Survival

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    <div><p>Bilirubin is the terminal metabolite in heme catabolism in mammals. After deposition into bile, bilirubin is released in large quantities into the mammalian gastrointestinal (GI) tract. We hypothesized that intestinal bilirubin may modulate the function of enteric bacteria. To test this hypothesis, we investigated the effect of bilirubin on two enteric pathogens; enterohemorrhagic <i>E. coli</i> (EHEC), a Gram-negative that causes life-threatening intestinal infections, and <i>E. faecalis</i>, a Gram-positive human commensal bacterium known to be an opportunistic pathogen with broad-spectrum antibiotic resistance. We demonstrate that bilirubin can protect EHEC from exogenous and host-generated reactive oxygen species (ROS) through the absorption of free radicals. In contrast, <i>E. faecalis</i> was highly susceptible to bilirubin, which causes significant membrane disruption and uncoupling of respiratory metabolism in this bacterium. Interestingly, similar results were observed for other Gram-positive bacteria, including <i>B. cereus</i> and <i>S. aureus</i>. A model is proposed whereby bilirubin places distinct selective pressure on enteric bacteria, with Gram-negative bacteria being protected from ROS (positive outcome) and Gram-positive bacteria being susceptible to membrane disruption (negative outcome). This work suggests bilirubin has differential but biologically relevant effects on bacteria and justifies additional efforts to determine the role of this neglected waste catabolite in disease processes, including animal models.</p></div

    <i>E. faecalis</i> metabolism decreases after exposure to bilirubin.

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    <p>(A) <i>E. coli</i> (86-24), <i>E. faecalis</i>, <i>B. cereus</i>, and <i>S. aureus</i> were supplemented with resazurin and incubated with heme (50 µM), biliverdin (500 µM), bilirubin (50 µM), and bilirubin ditaurate (500 µM) for either 30 minutes or 2 hours. Unreduced resazurin was monitored by absorbance at 600 nm. (B) <i>E. faecalis</i> cultures were supplemented with resazurin and solvent (NaOH, Sol.), bilirubin (100 µM, BR), biliverdin (100 µM, BV), or TTF (1 mM, a known inhibitor of succinate dehydrogenase) while incubated in 1× PBS with 0.5% sucrose for 30 minutes at 37°C. (C) Similar to panel B, <i>E. faecalis</i> cultures were supplemented with resazurin and either superoxide dismutase (SOD, 1000 U/mL) or heat-inactivated superoxide dismutase (SODi, 1000 U/mL). (D) <i>E. faecalis</i> supplemented with resazurin and diluted into 1× PBS with 0.5% sucrose (+Suc.) or without sucrose (−Suc.) and incubated for 30 minutes at 37°C. (E) <i>E. faecalis</i> supplemented with resazurin and increasing amounts of bilirubin (1, 5, 10, 20, 30, 50, and 100 µM) in similar conditions as panels B and C. Error bars represent ± one standard deviation, n = 3, and the (*) denotes a significant (P≤0.05) difference while (**) denotes a non-significant difference (P>0.05) between treated samples and solvent-treated samples.</p

    The effect of bile on EHEC growth in the presence of ROS.

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    <p>(A, Left) Wideband absorbance (420–580 nm) of EHEC (EDL933) cultures supplemented with plumbagin (0, 25, 50, and 75 µM) was monitored while cultures were grown at 37°C with shaking. (A, Right) The time to mid-log phase of each culture was calculated from the growth curves. (B) EHEC (EDL933) cultures supplemented with (grey bars) or without (white bars) plumbagin (50 µM) and/or BSA (2, 20, and 200 uM BSA) and/or ox bile (50, 100, 500, 1000 ug/mL ox bile). (C) EHEC (86-24) cultures were supplemented with plumbagin (50 µM) (grey bars) or without plumbagin (white bars) and either ox, rabbit (Rb), or human (Hu) bile (1 and 10 mg/mL ox bile; 0.5 and 5.0% rabbit and human bile). Error bars represent ± one standard deviation, n = 3, and (*) denotes a significant (P≤0.05) difference between treated samples and solvent-treated samples.</p

    Bilirubin protects EHEC from killing by J774A.1 macrophages.

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    <p>EHEC (86-24) was cultured with solvent (white), bilirubin (orange, 250 µM), biliverdin (green, 250 µM) or α-tocopherol (blue, 250 µM), in minimal media for 6 hours before addition to macrophages (J774A.1) at an MOI of approximately 3. (A) The amount of EHEC exposed to the macrophages is compared to the amount internalized by macrophages after 30 minutes. (B) The percentage of internalized EHEC over 2 hours was monitored for bacteria cultured with solvent (white diamonds, NaOH), bilirubin (orange squares), biliverdin (green circles) and α-tocopherol (blue triangles). Error bars represent ± one standard deviation, n = 3. Data are representative of a single experiment repeated three times with similar results and the (*) denotes a significant (P≤0.05) difference while (**) denotes a non-significant difference (P>0.05) between treated samples and solvent-treated samples.</p

    Fold reduction in viability of bacteria exposed to bilirubin.

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    <p>Mid-log phase bacteria were exposed to 200 µM bilirubin and plated to determine CFUs. Fold reduction was calculated by the quantity of bacteria unexposed to bilirubin divided by the quantity present when exposed to bilirubin, n = 3.</p

    Membrane disruption by bilirubin.

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    <p>(A) Bacteria including EHEC (86-24), <i>E. faecalis</i>, <i>S. aureus</i>, and <i>B. cereus</i> were incubated with heme, biliverdin, bilirubin, and bilirubin ditaurate (0, 1, 5, 10, 20, 50, 75, 100, 250 and 500 µM for heme and bilirubin (B,C), or 500 µM for biliverdin and bilirubin ditaurate (A)) and membrane permeability monitored by propidium iodide fluorescence. (D) <i>E. faecalis</i> OG1RF was cultured to mid-log phase and exposed to bilirubin, alpha-tocopherol, and CCCP (each 100 µM). DiSC<sub>3</sub>(5) (1 µM final concentration), a fluorescent compound which increases in intensity when associated with polarized membranes, was supplemented into cultures before quantifying the fluorescence intensity at excitation 622 nm and emission 670 nm. Error bars represent ± one standard deviation, n = 3, and (*) denotes a significant (P≤0.05) difference while (**) denotes a non-significant difference (P>0.05) between treated samples and solvent-treated samples.</p

    A Community-Based Nutrition and Physical Activity Intervention for Children Who Are Overweight or Obese and Their Caregivers

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    There is a need for efficacious interventions to reduce the prevalence of childhood obesity, and a limited body of research suggests that collaborative community-based programs designed for children and their caregivers may be effective in reducing obesity rates. This paper reports the results of a community-based obesity intervention, South County Food, Fitness and Fun (SCFFF), designed for preadolescent children who are overweight or obese and their caregivers. SCFFF was developed in response to community concerns. Families were referred to the program by their physician and participated in the program at no cost. The 16-week intervention includes weekly group nutrition and physical activity sessions. Analyses determined that 65 out of the 97 children who completed SCFFF provided 2-year follow-up data and had reduced BMI z-scores over 2 years following the intervention. These participants decreased their energy, fat, carbohydrate, saturated fat, and sodium intake and increased core body strength and endurance from baseline to the end of the intervention. SCFFF was effective in reducing relative weight and improving diet and core muscle strength and endurance in children who are overweight or obese
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