This Loc-Carrillo_README-20170817.txt file was generated on [20170817] by [Catherine Loc-Carrillo] Links to Publication Field updated 20211209 DN ------------------- GENERAL INFORMATION ------------------- 1. Title of Dataset: Pharmacokinetics of Locally Delivered Vancomycin to Bone 2. Author Information: Catherine Loc-Carrillo, Caroline Wang, Ahranee Canden, Michael Burr, Jayant Agarwal Principal Investigator Contact Information Name: Jayant Agarwal Institution: VA Salt Lake City Health Care System; University of Utah School of Medicine Address: VA Salt Lake City Health Care System, Salt Lake City, Utah, United States of America; Division of Plastic and Reconstructive Surgery, Department of Surgery, University of Utah School of Medicine, Salt Lake City, Utah, United States of America Email: jay.agarwal@hsc.utah.edu Associate or Co-investigator Contact Information Name: Catherine Loc-Carrillo Institution: VA Salt Lake City Health Care System; University of Utah School of Medicine Address: VA Salt Lake City Health Care System, Salt Lake City, Utah, United States of America; Division of Epidemiology, Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, Utah, United States of America Email: c.loc.carrillo@hsc.utah.edu Alternate Contact Information Name: Institution: Address: Email: 3. Date of data collection (single date, range, approximate date) <20130501 - 20150130> 4. Geographic location of data collection (where was data collected?): Salt Lake City, Utah 5. Information about funding sources that supported the collection of the data: This work was supported by the University of Utah Research Foundation to Jay Agarwal. -------------------------- SHARING/ACCESS INFORMATION -------------------------- 1. Licenses/restrictions placed on the data: None. 2. Links to publications that cite or use the data: Loc-Carrillo C, Wang C, Canden A, Burr M, Agarwal J (2016) Local Intramedullary Delivery of Vancomycin Can Prevent the Development of Long Bone Staphylococcus aureus Infection. PLoS ONE 11(7): e0160187. https://doi.org/10.1371/journal.pone.0160187 3. Links to other publicly accessible locations of the data: PLoSONE https://doi.org/10.1371/journal.pone.0160187 4. Links/relationships to ancillary data sets: None 5. Was data derived from another source? No If yes, list source(s): 6. Recommended citation for the data: Catherine Loc-Carrillo, Caroline Wang, Ahranee Canden, Michael Burr, Jayant Agarwal. 2017. Pharmacokinetics of Locally Delivered Vancomycin to Bone. The Hive: University of Utah Research Data Repository. https://doi.org/10.7278/S5W0942B --------------------- DATA & FILE OVERVIEW --------------------- 1. File List A. Filename: Bacterial innoculumns Short description: Bacterial load delivered to each rat that underwent the prevention surgery. B. Filename: HPLC analysis Short description: HPLC analysis of detectable vancomycin concentration in tibia, muscle, and plasma after intramedullary delivery in each rat. C. Filename: IVIS analysis Short description: Bioluminescent activity detected by IVIS for each rat that underwent prevention study. 2. Relationship between files: All three files contain data for the same set of rats used in our prevention study. 3. Additional related data collected that was not included in the current data package: X-ray, IVIS, and micro-CT images of each rat were also collected but could not be included in this repository. 4. Are there multiple versions of the dataset? No If yes, list versions: Name of file that was updated: i. Why was the file updated? ii. When was the file updated? Name of file that was updated: i. Why was the file updated? ii. When was the file updated? -------------------------- METHODOLOGICAL INFORMATION -------------------------- 1. Description of methods used for collection/generation of data: ANIMALS: Male Sprague-Dawley rats (Charles River Lab Inc. Wilmington, MA) > 9 weeks old and > 500 grams in weight were housed in a controlled environment with regulated temperature, 12-h light/dark cycle, and were allowed food and water ad libitum. The Salt Lake City VA’s Institutional Animal Care and Use Committee (IACUC) approved the animal use and experimental protocol, prior to commencing the study (ACORP 13–01). Rats were prepared for surgery by washing tail and shaving fur from the lower half of their body using an electric clipper, with a 0.25 mm blade (40SS CeramicEdge, Andis, Sturtevant, WI.). Immediately prior to surgery, the feet, legs, groin and abdomen were scrubbed with chlorhexidine soap and prepped with chlorhexidine clear solution. All surgery was performed under a mixture of isoflurane and oxygen anesthesia, and all efforts were made to minimize suffering. IN-VIVO PHARMACOKINETICS: Rats (n = 4 per time-point) were used to evaluate the local (bone, muscle) and systemic (plasma) concentration of vancomycin after local intramedullary delivery to the rat tibia. While under general anesthesia, the right lower extremity was passed through a sterile half-drape and positioned away from the surgeon. A longitudinal incision was made with a sterile scalpel (#10), in the sagittal plane, extending 10 mm distally from the tibial tuberosity. A sterile sharp-ended Kirschner wire (0.054 in diameter) was used, at the proximal fourth junction, to make a hole through the cortical bone into the medial metaphysis and medullary canal of the tibia. The medullary cavity was then gently reamed with a 21G 1 1/2” needle, followed by reaming with an 18G 1” needle in order to reach the distal part of the tibial canal. The cavity of the medullary canal was excavated as much has possible by attaching a sterile syringe to the end of the 18G 1” needle and aspirating the contents 3–4 times before applying treatment. Gentle reaming provided clearance of intramedullary tissue/debris to allow for uniform filling of the medullary canal. Vancomycin hydrochloride solution (i.e., ~50 μL; 25 mg/Kg dose) was then injected into the canal using a sterile 21G needle and Hamilton syringe. The hole was then sealed with sterile bone wax and the incision was closed using stainless steel wound clips or simple stiches. Buprenorphine (i.e., 0.01–0.05 mg/Kg) was administered for analgesia upon emergence from anesthesia. Rats were given food and water ad libitum and monitored daily until defined time-points (i.e., 0 h, 4 h, 24 h, 48 h, 72 h, and 96 h) when they were sacrificed. Before sacrificing the rats, 800 μL of blood was drawn from their tail vein. At sacrifice, the tibia, and surrounding muscles were harvested and prepared for HPLC analysis. It should be noted that the medullary canal was reamed to remove any contents prior to analyzing the bone concentration. Therefore the bone concentration is a true representation of drug within the bone. HPLC ANALYSIS: High performance liquid chromatography (HPLC) was used to detect vancomycin in bone, muscle, and plasma samples. For all tissue samples an Acclaim 120 C18 reverse phase column with 5μm particle diameter, 4.6 inner column diameter, and 250 mm in length (Thermo Fisher). Detection was done at 235 nm with an Agilent Technologies 1260 Infinity diode array detector at a temperature of 45°C. Bone extractions were done by adding 1% formic acid at 0.5g/mL to pulverized tissue. Samples were vortexed for 1 min and then centrifuged at 12,000 rpm for 10 min. The supernatant was injected after filtration with a 0.22 μm PES filter. Muscle extractions were done by adding mobile phase at 1.0 g/mL to pulverized tissue. Samples were vortexed for 1 min and then centrifuged at 15,000 rpm for 10 min. The supernatant was injected after filtration with a 0.22 μm PES filter. Plasma samples were diluted in mobile phase at a 1:1 ratio and injected as such. Calibration curves were done for each tissue type with blank matrix. Levels below 1 μg/mL were not detectable. All area under the curve (AUC) measurements were determined using Chemstation software Rev. B.04.03 (Agile Technologies, Santa Clara, CA). The mobile phase was a premixed solution of 90:10 50mM KH2PO4 in 0.1% formic acid:acetonitrile. Adjustments were made with 0.1% formic acid in acetonitrile based on tissue type as needed. PREPARATION OF BACTERIAL INOCULUM: Bioluminescent strain Staphylococcus aureus Xen 36 (Caliper LifeSciences, Alameda, CA) was used to develop bone infection. The strain was stored in a glycerol (20% v/v) and Brain Heart Infusion (BHI) suspension at -80°C until required. Bacteria were subcultured onto an LB plate (Luria-Bertani, Miller broth mixed with 1.2% (w/v) Technical Agar (Difco, Sparks, MD.)), at 37°C overnight. Five colonies were then transferred to 3 mL LB broth and grown in shaker incubator (200 rpm), at 37°C for 2.5 h. Two milliliters of the bacterial suspension was transferred to 100 mL LB broth and grown for a further 4 h, until the cells reached exponential growth. Bacterial suspension was transferred into 50 mL centrifuge tubes and cells centrifuged at ~3,000 rpm for 30 min. Supernatants were discarded and pellets were re-suspended in 15 mL Phosphate Buffered Saline (PBS). Cells were then mixed by vortexing and the centrifugation process was repeated. This cell washing process was repeated three times. The final washed and pelleted cells were re-suspended in a total of 10 mL PBS, and mixed by vortexing. The cell suspension was then ten-fold serially diluted. The concentration of the cell suspension was then determined using the spread plate technique and spreading 100 μL of the various dilutions onto LB plates, in duplicate. Plates were incubated at 37°C overnight before counting colony forming units (CFU). The viable count was then used to dilute the washed cell suspension to the desired concentrations (i.e., average Log10 7.82 CFU/mL or 6.62x107 CFU/mL) using PBS. Two hundred microliter aliquots were dispensed into microfuge tubes and labeled bacterial inoculum. To inoculate the rat tibias the bacterial inoculum was mixed with equal volume of 1% (w/v) molten agarose (Pulsed field certified agarose, Biorad, Hercules, CA). Mixing the bacteria with agarose helped to control seepage of the inoculum and minimize contamination of the surrounding muscle. IN-VIVO INFECTION PREVENTION STUDY: While under general anesthesia, a corticotomy was made in the tibia as described above, and 10 μL bacterial inoculum (i.e., average Log10 7.82 CFU/mL in 0.5% w/v agarose) was added to medullary canal followed by vancomycin solution (i.e., ~ 50 μL; 25mg/Kg dose). The hole was then sealed with sterile bone wax (Sharpoint™, Surgical Specialties Corp.) and the wound was closed using 7 mm stainless steel wound clips (Reflex™ Skin Closure System, CellPoint Scientific Inc., Gaitherburg, MD) or simple stitches (S&T Microsurgical Suture w/suture thread, Fine Science Tools, Foster City, CA). IN-VIVO IMAGING: Images of the rats were taken using the IVIS Lumina II Imaging System (Caliper Life Science, Alameda, CA). Rats were sedated using isoflurane (1–5%) and oxygen (2 L/min) mixture during imaging. Rats were placed in the supine position inside the cabinet and placed with their right hind leg in the center of the field. The following parameters were used when taking the images: filed of view (FOV) = 12.5; binning = 8; f-stop = 1.0; and exposure time = 300 s. The luminescent color scale for all IVIS images was normalized to have a min and max radiance range of 3.48e4 and 3.48e5 (p/sec/cm2/sr), respectively. MICROBIOLOGICAL ANALYSIS: After sacrificing the rats, the tibia and gastrocnemius muscle surrounding the surgical site were explanted, weighed separately and flash frozen with liquid nitrogen prior to pulverizing in a freezer mill (6770 Spex SamplePrep, Metuchen, NJ.). The Spex SamplePrep parameters used were: two cycles of pre-cool- 5 mins; run time- 5 mins; cool time- 2 min; rate- 10 CPS. The powdered samples were then reweighed, suspended (i.e., 1:10) in phosphate buffered saline (PBS), vortexed, and 10-fold serially diluted before plating (in duplicate) 100 μL of suspensions onto LB plates (Luria-Bertani, Miller broth mixed with 1.2% (w/v) Technical Agar (Difco, Sparks, MD.)). Plates were incubated at 37°C for 18–24 h and the bacterial load (colony forming units) per sample was calculated based on the number of colonies seen at specific dilutions. Confirmation of isolating S. aureus Xen 36 colonies from samples was determined using the IVIS (Lumina II, Caliper Life Science, Hopkinton, MA.). Non-bioluminescent colonies were confirmed as S. aureus using Gram staining, catalase test and Color Staph ID kit (Sure-Vue, Fisher Scientific, Houston TX.). 2. Methods for processing the data: The following parameters were used to obtain the photon counts when taking the IVIS images: field of view (FOV) = 12.5; binning = 8; f-stop = 1.0; and exposure time = 300 s. 3. Instrument- or software-specific information needed to interpret the data: The IVIS data were obtained using the Living Image Software, version 4.2 (Caliper Life Sciences). 4. Standards and calibration information, if appropriate: For the HPLC analysis, calibration curves were done by spiking known quantities of drug in blank matrix extracts followed by serial dilutions. The curve was linear in the range quantified from 1 to 16 μg/mL. For the IVIS analysis, the photon counts obtained for the region of interest were standardized to have a min and max radiance range of 150 and 1500, respectively. 5. Environmental/experimental conditions: None. 6. Describe any quality-assurance procedures performed on the data: Instruments and software were maintained under the manufacturer’s service agreement. 7. People involved with sample collection, processing, analysis and/or submission: Sample collection: Catherine Loc-Carrillo, Caroline Wang, Sheena Fernandez, Sijia Wu, Kelly Hoerger, John Churchill and Hunter Fredricksen. Sample processing: Catherine Loc-Carrillo, Caroline Wang, Michael Burr. Sample analysis: Catherine Loc-Carrillo, Jay Agarwal Submission: Catherine Loc-Carrillo ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: [Bacterial innoculumns] ----------------------------------------- 1. Number of variables: 2. Number of cases/rows: 3. Variable List A. Name: [variable name] Description: [description of the variable] Value labels if appropriate B. Name: [variable name] Description: [description of the variable] Value labels if appropriate ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: [HPLC analysis] ----------------------------------------- 1. Number of variables: 2. Number of cases/rows: 3. Variable List A. Name: [variable name] Description: [description of the variable] Value labels if appropriate B. Name: [variable name] Description: [description of the variable] Value labels if appropriate ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: [IVIS analysis] ----------------------------------------- 1. Number of variables: 2. Number of cases/rows: 3. Variable List A. Name: [variable name] Description: [description of the variable] Value labels if appropriate B. Name: [variable name] Description: [description of the variable] Value labels if appropriate 4. List of abbreviations used: AUC - area under the curve AVE - average CFU - colony forming units DF - dilution factor g - grams HPLC - high performance liquid chromatography hr - hours Kg - kilograms MIC - minimum inhibitory concentration mL - milliliters MRSA - methicillin-resistant staphylococcus aureus SE - standard error STDEV - standard deviation ug - microliters