Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Animal Sciences
Non Technical Summary
Extra-intestinal pathogenic Escherichia coli (ExPEC) gut colonization, although asymptomatic, increases the risk of subsequent extra-intestinal infections, including urinary tract infection and septicemia. Current studies indicate that poultry, beef and pork products may be a significant source of ExPEC and that 20-45% of ExPEC are now resistant to first line antibiotics. Our previous work shows natural biopolymers such as tannins or proanthocyanidins (PAC), inhibit ExPEC adhesion and reduce invasion of enterocytes. We have invented novel composite biomaterials comprising formulations of PAC extracted from cranberries and chitosan manufactured from shrimp exoskeletons. These tannin-chitosan composite nanoparticles are being evaluated as viable alternatives to antibiotic therapy and may help stem the alarming increase in drug resistant bacteria. The goals of this proposal are to gain a better understanding of how: 1) tannin-chitosan formulations (tannin source, ratios and chemical structure) can be manipulated to increase specificity and efficacy against ExPEC and 2) how tannin-chitosan composite deliverables can be manipulated to meet current and future applications (alternatives to antibiotics). Results of these studies will support the "sustainable use of natural resources" (cranberry fruit and process waste streams), address "food safety" issues and lead to new applications of natural products as alternatives to antibiotics.
Animal Health Component
50%
Research Effort Categories
Basic
(N/A)
Applied
50%
Developmental
50%
Goals / Objectives
The goals of this proposal are to: 1) gain a better understanding of how tannin-chitosan formulations (tannin source, ratios and chemical structure) can be manipulated to increase specificity and efficacy against ExPEC and 2) gain a better understanding of how tannin-chitosan composite deliverables can be manipulated (casting of foams, films, and hydrogels) to meet current and future applications.
Project Methods
1. Formulation of tannin-chitosan composite nanoparticles: 1.1) Extraction and preparation of c-PAC: Based on our knowledge of cranberry polyphenol chemistry15-17and prior experience relating structure of c-PAC to biological function,5, 18, 19we have selected cranberry fruit as our source material for the extraction of PAC. Cranberry fruit (variety 'Stephens') will be harvested from a single plot. Briefly, the fruit will be blended with liquid nitrogen to produce a fine homogeneous powder, exhaustively extracted with 70% aqueous acetone (v/v), centrifuged to remove particulate matter and concentrated under vacuum (<30°C) to remove the acetone. The aqueous solution will be applied to a Sephadex LH-20 column (equilibrated in ethanol). The column will be eluted sequentially with ethanol and 50% ethanol:methanol (v/v) to remove hydroxycinnamic acids, anthocyanins, and flavonols. The resin will then be eluted with 70% aqueous acetone (v/v) to recover c-PAC.1.2) Mass Spectrometry:MALDI-TOF/TOF MS and will be applied to characterize the molecular weight distribution,of c-PAC, unique structural features of the c-PAC fractions and to quantify A-type to B-type interflavan bonds.5, 16, 17, 20-24Each c-PAC fraction will be combined with the matrix (2,5-dihydroxybenzoic acid) and analyzed by two MS methods. MALDI TOF/TOF MS will be performed on a Bruker ULTRAFLEX®III MALDI TOF/TOF mass spectrometer (Billerica, MA, USA) equipped with delayed extraction and a SmartBeam® laser. Mass spectra will be calibrated with bradykinin and glucagon as external standards. All fractions will be analyzed in the positive ion mode using an accelerating voltage of 25.0 kV and a reflectron voltage of 26.25 kV.1.3) Synthesis of tannin-chitosan nanoparticlesPharmaceutical grade (>90% deacetlyation) chitosan will be solubilized in a 1.0% acetic acid solution at concentrations ranging from 5-15g/L. The stock chitosan solution will then be diluted with water (1- 15 mg/mL). PAC-chitoasn nanoparticles will be formulated by mixing 5ml of diluted chitosan solution with 5 ml (20-100mg /mL) PAC extract in a 10 ml flask. Solutions will be stirred vigorously with stir bar for 15 minutes. While maintaining a constant stirring rate, varying volumes of tripolyphosphate solution (1mg/mL) will be added quickly to each solution. Water will be added to bring the final volume to 7 mL. Solutions will be stirred for another 15 minutes. At the end of this period, nanoparticle size will be determined by methods of light refraction. The composites nanoparticles will be subsequently be used in biological assays described below.2. Inhibition of enterocyte invasion by pathogenic E. coli.2.1). Inhibition of E. coli invasion of Caco-2 cells assay:Caco-2 (HTB-37) cells will be cultured in DMEM supplemented with 10% FBS, 1% non-essential amino acids, 1% L-alanyl-L-glutamine, and 1% penicillin (100 units/mL) / streptomycin (100µg/mL) at 37ºC in 5% CO2. For use in experiments, cells will be seeded density that is 80% of confluency (1) into 24-well Transwell® permeable support plates (Corning, Lowell, MA) for cell invasion studies, (2) into 24-well MatTek culture treated plates (MatTek Corp., Ashland, MA) for fluorescence microscopy studies or (3) on sterile cover slips placed into 24-well plates for electron microscopy studies. Once the cells reach confluence, they will be allowed to differentiate for 20 days to become phenotypic polarized enterocytes. Bacterial Cells: ExPEC-5011, a clinical isolate from women with chronic urinary tract infection, which expresses both Type-1 and P Type Fimbriae, will be cryogenically maintained until three days prior to experiments. Prior to the experiment, the strains will be grown in tryptose broth containing Ampicillin at 37ºC under a series of static aerobic culturing conditions that enriches the expression of fimbriae.Differentiated Caco-2 cells will be incubated with washed and resuspended bacterial cells at the appropriate multiplicity of infection (MOI) for 3h at 37ºC. When assessing effects of PAC-chitosan nanoparticles on cell invasion, the microbial culture will be pre-incubated with nanoparticles for 5 minutes, washed and resuspended prior to the incubation with Caco-2 cells. To assess cell invasion: After the incubation period, the Caco-2 cells will be washed and treated with gentamicin sulphate (100 µg/mL) for 1h at 37ºC to kill extracellular ExPEC (adhered to the surface of the cells). Cells will then be washed twice and lysed with 1% Triton solution at room temperature. Dilutions of the cell lysate will be plated on Levine's eosin-methylene blue (EMB) + Ampicillin plates and incubated overnight at 37ºC. Colony forming units (CFUs) will be counted to enumerate invading UPEC. To assess cell adherence: After the incubation period, the Caco-2 cells will be washed (but not treated with gentamicin) and lysed with 1% Triton-X at room temperature (with agitation), plated and the ExPEC enumerated as before. Total adherent bacteria = number of bacterial cells adhered to extracellular surface of Caco-2 + number of intracellular (invaded) bacterial cells.2.2) Determination of ExPEC adherence to and invasion using EVISC model:Male (7 to 8 weeks old) Institute of Cancer Research Mice (ICR; Harlan, Indianapolis, IN) will be fed standard mouse chow (Rodent Diet 5001; LabDiet, PMI Nutrition International, St. Louis, MO) and water ad libitum for 1 week prior to initiation of study protocol. The mice will be anesthetized by intraperitoneal injection of ketamine (100 mg/kg BW) and acepromazine (10 mg/kg BW), and euthanized via exsanguination of the left axillary artery. The small intestine will be harvested, cleaned of mesenteric fat and vascular tissue. The lumen will be flushed with 60 mL of HBSS and then 60 mL of RPMI. 1.5 cm segments of distal ileum free of Peyer's patches will be isolated and carefully opened apical side up. Tissue glue (Dermabond, Ethicon, Cornelia, GA) will be lightly placed on one side of a plastic tissue disc (polystyrene with a 9 mm diameter and 6 mm internal aperture) and lowered over the intestinal segment. Once the glue sets (~10 seconds), the tissue disc and intestinal segment will be turned over (serosal side up) and sandwiched with a second tissue disc lightly covered with tissue glue. Once the second tissue disc is adhered, a light layer of tissue glue will be applied to the bottom of the serosal disc and lowered into a cell culture insert (Cat 3292, 3.0 µM pore, 12 well format, BD bioscience, NJ). Gentle pressure will be applied to ensure adherence of the bottom tissue disc to the cell culture insert. Schematic of tissue discs, intestinal segment, and cell culture insert are shown at right. Cell culture inserts will be placed into 12 well plates prefilled with 1 mL RPMI+Amp per well. The intestinal segments will be incubated with 400 µL of washed and resuspended bacterial cells (alone or after treatment with PAC-chitosan nanoparticles as described in section 2.2) at 1 x 108 CFU/mL RPMI+Amp for 1h at 37ºC. The wells will be rinsed thrice with DPBS. Then gentamicin will be added to each well for 1 hour at 37ºC. This will kill any remaining bacteria in the well or adhered to the mucosal surface. The tissue segment will be rinsed and lysed with 1% Triton-X at rm. temp (with agitation). Enumeration of cell invasion and cell adherence will performed as before in Caco-2 cell experiments.